Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii

ABSTRACT

The present application describes the complete 1.66-megabase pair genome sequence of an autotrophic archaeon,  Methanococcus jannaschii , and its 58- and 16-kilobase pair extrachromosomal elements. Also described are 1738 predicted protein-coding genes.

This application is a Continuation of U.S. application Ser. No. 08/916,421, filed Aug. 22, 1997, which is hereby incorporated by reference; said U.S. application Ser. No. 08/916,421 claims priority of U.S. Provisional Application No. 60/024,428, filed Aug. 22, 1996, which is hereby incorporated by reference.

Part of the work performed during development of this invention utilized U.S. Government funds. The U.S. Government may have certain rights in the invention—DE-FC02-95ER61962; DE-FCO2-95ER61963; and NAGW 2554.

BACKGROUND OF THE INVENTION Statement as to Rights to Inventions Made Under Federally-Sponsored Research and Development

1. Reference to a Sequence Listing Provided on Compact Disc

This application refers to a “Sequence Listing”, which is provided as an electronic document on two identical compact discs (CD-R), labeled “Copy 1” and “Copy 2.” These compact discs each contain the electronic document, filename “PB275C1.ST25.txt” (2,259,883 bytes in size, created on Nov. 14, 2002), which is hereby incorporated in its entirety herein.

2. Field of the Invention

The present application discloses the complete 1.66-megabase pair genome sequence of an autotrophic archaeon, Methanococcus jannaschii, and its 58- and 16-kilobase pair extrachromosomal elements. Also identified are 1738 predicted protein-coding genes.

3. Related Background Art

The view of evolution in which all cellular organisms are in the first instance either prokaryotic or eukaryotic was challenged in 1977 by the finding that on the molecular level life comprises three primary groupings (Fox, G. E., et al., Proc. Natl. Acad. Sci. USA 74:4537 (1977); Woese, C. R. & Fox, G. E., Proc. Natl. Acad. Sci. USA 74:5088 (1977); Woese, C. R., et al., Proc. Natl., Acad. Sci. USA 87:4576 (1990)): the eukaryotes (Eukarya) and two unrelated groups of prokaryotes, Bacteria and a new group now called the Archaea. Although Bacteria and Archaea are both prokaryotes in a cytological sense, they differ profoundly in their molecular makeup (Fox, G. E., et al., Proc. Natl. Acad. Sci. USA 74:4537 (1977); Woese, C. R. & Fox, G. E., Proc. Natl. Acad. Sci. USA 74:5088 (1977); Woese, C. R., et al., Proc. Natl. Acad. Sci. USA 87:4576 (1990)). Several lines of molecular evidence even suggest a specific relationship between Archaea and Eukarya (Iwabe, N., et al., Proc. Natl. Acad. Sci. USA 86:9355 (1989); Gogarten J. P., et al., Proc. Natl. Acad. Sci. USA 86:6661 (1989); Brown, J. R. and Doolittle, W. F., Proc. Natl. Acad. Sci. USA 92:2441 (1995)).

The era of true comparative genomics has been ushered in by complete genome sequencing and analysis. We recently described the first two complete bacterial genome sequences, those of Haemophilus influenzae and Mycoplasma genitalium (Fleischmann, R. D., et al., Science 269:496 (1995); Fraser, C. M., et al., Science 270:397 (1995)). Large scale DNA sequencing efforts also have produced an extensive collection of sequence data from eukaryotes, including Homo sapiens (Adams, M. D., et al., Nature 377:3 (1995)) and Saccharomyces cerevisiae (Levy, J., Yeast 10:1689 (1994)).

M. jannaschii was originally isolated by J. A. Leigh from a sediment sample collected from the sea floor surface at the base of a 2600 m deep “white smoker” chimney located at 21° N on the East Pacific Rise (Jones, W., et al., Arch. Microbiol. 136:254 (1983)). M. jannaschii grows at pressures of up to more than 500 atm and over a temperature range of 48-94° C. with an optimum temperature near 85° C. (Jones, W., et al., Arch. Microbiol. 136:254 (1983)). The organism is autotrophic and a strict anaerobe; and, as the name implies, it produces methane. The dearth of archaeal nucleotide sequence data has hampered attempts to begin constructing a comprehensive comparative evolutionary framework for assessing the molecular basis of the origin and diversification of cellular life.

SUMMARY OF THE INVENTION

The present invention is based on whole-genome random sequencing of an autotrophic archaeon, Methanococcus jannaschii. The M. jannaschii genome consists of three physically distinct elements: (i) a large circular chromosome; (ii) a large circular extrachromosomal element (ECE); and (iii) a small circular extrachromosomal element (ECE). The nucleotide sequences generated, the M. jannaschii chromosome, the large ECE, and the small ECE, are respectively provided on pages 153-586 (SEQ ID NO:1), pages 586-601 (SEQ ID NO:2), and pages 602-606 (SEQ ID NO:3).

The present invention is further directed to isolated nucleic acid molecules comprising open reading frames (ORFs) encoding M. jannaschii proteins. The present invention also relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of M. jannaschii proteins. Further embodiments include isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to the nucleotide sequence of a M. jannaschii ORF described herein.

The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, host cells containing the recombinant vectors, as well as methods for making such vectors and host cells for M. jannaschii protein production by recombinant techniques.

The invention further provides isolated polypeptides encoded by the M. jannaschii ORFs. It will be recognized that some amino acid sequences of the polypeptides described herein can be varied without significant effect on the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity. In general, it is possible to replace residues which form the tertiary structure, provided that residues performing a similar function are used. In other instances, the type of residue may be completely unimportant if the alteration occurs at a non-critical region of the protein.

In another aspect, the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention. The epitope-bearing portion is an immunogenic or antigenic epitope useful for raising antibodies.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. A schematic showing the relationship of the three domains of life based on sequence data from the small subunit of rRNA (Fox, G. E., et al., Proc. Natl. Acad. Sci. USA 74:4537 (1977); Woese, C. R. & Fox, G. E., Proc. Natl. Acad. Sci. USA 74:5088 (1977); Woese, C. R., et al., Proc. Natl. Acad. Sci. USA 87:4576 (1990)).

FIG. 2. Block diagram of a computer system 102 that can be used to implement the computer-based systems of present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on whole-genome random sequencing of an autotrophic archaeon, Methanococcus jannaschii. The M. jannaschii genome consists of three physically distinct elements: (i) a large circular chromosome of 1,664,976 base pairs (bp) (shown on pages 153-586 and in SEQ ID NO:1), which contains 1682 predicted protein-coding regions and has a G+C content of 31.4%; (ii) a large circular extrachromosomal element (the large ECE) of 58,407 bp (shown on pages 586-601 and in SEQ ID NO:2), which contains 44 predicted protein-coding regions and has a G+C content of 28.2%; and (iii) a small circular extrachromosomal element (the small ECE) of 16,550 bp (shown on pages 602-606 and in SEQ ID NO:3), which contains 12 predicted protein-coding regions and has a G+C content of 28.8%.

The primary nucleotide sequences generated, the M. jannaschii chromosome, the large ECE, and the small ECE, are provided in SEQ ID NOs:1, 2, and 3, respectively. As used herein, the “primary sequence” refers to the nucleotide sequence represented by the IUPAC nomenclature system. The present invention provides the nucleotide sequences of SEQ ID NOs:1, 2, and 3, or a representative fragment thereof, in a form which can be readily used, analyzed, and interpreted by a skilled artisan.

As used herein; a “representative fragment” refers to M. jannaschii protein-encoding regions (also referred to herein as open reading frames), expression modulating fragments, uptake modulating fragments, and fragments that can be used to diagnose the presence of M. jannaschii in a sample. A non-limiting identification of such representative fragments is provided in Tables 2(a) and 3. As described in detail below, representative fragments of the present invention further include nucleic acid molecules having a nucleotide sequence at least 90% identical, preferably at least 95, 96%, 97%, 98%, or 99% identical, to an ORF identified in Table 2(a) or 3.

As indicated above, the nucleotide sequence information provided in SEQ ID NOs:1, 2 and 3 was obtained by sequencing the M. jannaschii genome using a megabase shotgun sequencing method. The sequences provided in SEQ ID NOs:1, 2 and 3 are highly accurate, although not necessarily a 100% perfect, representation of the nucleotide sequence of the M. jannaschii genome. As discussed in detail below, using the information provided in SEQ ID NOs:1, 2 and 3 and in Tables 2(a) and 3 together with routine cloning and sequencing methods, one of ordinary skill in the art would be able to clone and sequence all “representative fragments” of interest including open reading frames (ORFs) encoding a large variety of M. jannaschii proteins. In rare instances, this may reveal a nucleotide sequence error present in the nucleotide sequences disclosed in SEQ ID NOs:1, 2, and 3. Thus, once the present invention is made available (i.e., once the information in SEQ ID NOs:1, 2, and 3 and in Tables 2(a) and 3 have been made available), resolving a rare sequencing error would be well within the skill of the art. Nucleotide sequence editing software is publicly available. For example, Applied Biosystem's (AB) AutoAssembler™ can be used as an aid during visual inspection of nucleotide sequences.

Even if all of the rare sequencing errors were corrected, it is predicted that the resulting nucleotide sequences would still be at least about 99.9% identical to the reference nucleotide sequences in SEQ ID NOs:1, 2, and 3. Thus, the present invention further provides nucleotide sequences that are at least 99.9% identical to the nucleotide sequence of SEQ ID NO:1, 2, or 3 in a form which can be readily used; analyzed and interpreted by the skilled artisan. Methods for determining whether a nucleotide sequence is at least 99.9% identical to a reference nucleotide sequence of the present invention are described below.

Nucleic Acid Molecules

The present invention is directed to isolated nucleic acid fragments of the M. jannaschii genome. Such fragments include, but are not limited to, nucleic acid molecules encoding polypeptides (hereinafter open reading frames (ORFs)), nucleic acid molecules that modulate the expression of an operably linked ORF (hereinafter expression modulating fragments (EMFs)), nucleic acid molecules that mediate the uptake of a linked DNA fragment into a cell (hereinafter uptake modulating fragments (UMFs)), and nucleic acid molecules that can be used to diagnose the presence of M. jannaschii in a sample (hereinafter diagnostic fragments (DFs)).

By “isolated nucleic acid molecule(s)” is intended a nucleic acid molecule, DNA or RNA, that has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells, purified (partially or substantially) DNA molecules in solution, and nucleic acid molecules produced synthetically. Isolated RNA molecules include in vitro RNA transcripts of the DNA molecules of the present invention.

In one embodiment, M. jannaschii DNA can be mechanically sheared to produce fragments about 15-20 kb in length, which can be used to generate a M. jannaschii DNA library by insertion into lambda clones as described in Example 1 below. Primers flanking an ORF described in Table 2(a) or 3 can then be generated using the nucleotide sequence information provided in SEQ ID NO:1, 2, or 3. The polymerase chain reaction (PCR) is then used to amplify and isolate the ORF from the lambda DNA library. PCR cloning is well known in the art. Thus, given SEQ ID NOs:1, 2, and 3, and Tables 2(a) and 3, it would be routine to isolate any ORF or other representative fragment of the M. jannaschi genome. Isolated nucleic acid molecules of the present invention include, but are not limited to, single stranded and double stranded DNA, and single stranded RNA, and complements thereof.

Tables 2(a), 2(b) and 3 describe ORFs in the M. jannaschii genome. In particular, Table 2(a) (pages 68-116 below) indicates the location of ORFs (i.e., the position) within the M. jannaschii genome that putatively encode the recited protein based on homology matching with protein sequences from the organism appearing in parentheticals (see the fourth column of Table 2(a)). The first column of Table 2(a) provides a name for each ORF. The second and third columns in Table 2(a) indicate an ORF's position in the nucleotide sequence provided in SEQ ID NO:1, 2 or 3. One of ordinary skill in the art will appreciate that the ORFs may be oriented in opposite directions in the M. jannaschii genome. This is reflected in columns 2 and 3. The fifth column of Table 2(a) indicates the percent identity of the protein sequence encoded by an ORF to the corresponding protein sequence from the organism appearing in parentheticals in the fourth column. The sixth column of Table 2(a) indicates the percent similarity of the protein sequence encoded by an ORF to the corresponding protein sequence from the organism appearing in parentheticals in the fourth column. The concepts of percent identity and percent similarity of two polypeptide sequences are well understood in the art and are described in more detail below. The eighth column in Table 2(a) indicates the length of the ORF in nucleotides. Each identified gene has been assigned a putative cellular role category adapted from Riley (Riley, M., Microbiol. Rev. 57:862 (1993)).

Table 2(b) (page 117 below) provides the single ORF identified by the present inventors that matches a previously published M. jannaschii gene. In particular, ORF MJ0479, which is 585 nucleotides in length and is positioned at nucleotides 1,050,508 to 1,049,948 in SEQ ID NO:1, shares 100% identity to the previously published M. jannaschii adenylate kinase gene.

Table 3 (pages 118-151 below) provides ORFs of the M. jannaschii genome that did not elicit a homology match with a known sequence from either M. jannaschii or another organism. As above, the first column in Table 3 provides the ORF name and the second and third columns indicate an ORF's position in SEQ ID NO:1, 2, or 3.

Table 4 (page 152 below) provides genes of M. jannaschii that contain inteins.

In the above-described Tables, there are three groups of ORF names. The one thousand six hundred and eighty two ORFs named “MJ-” (MJ0001-MJ1682) were identified on the M. jannaschii chromosome (SEQ ID NO:1). The forty four ORFs named “MJECL-” (MJECL01-MJECL44) were identified on the large ECE (SEQ ID NO:2). The twelve ORFs named “MJECS-” (MJECS01-MJES12) were identified on the small ECE (SEQ ID NO:3).

Further details concerning the algorithms and criteria used for homology searches are provided in the Examples below. A skilled artisan can readily identify ORFs in the Methanococcus jannaschii genome other than those listed in Tables 2(a), 2(b) and 3, such as ORFs that are overlapping or encoded by the opposite strand of an identified ORF in addition to those ascertainable using the computer-based systems of the present invention.

Isolated nucleic acid molecules of the present invention include DNA molecules having a nucleotide sequence substantially different than the nucleotide sequence of an ORF described in Table 2(a) or 3, but which, due to the degeneracy of the genetic code, still encode a M. jannaschii protein. The genetic code is well known in the art. Thus, it would be routine to generate such degenerate variants.

The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of a M. Jannaschii protein encoded by an ORF described in Table 2(a) or 3. Non-naturally occurring variants may be produced using art-known mutagenesis techniques and include those produced by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the M. jannaschii protein or portions thereof. Also especially preferred in this regard are conservative substitutions.

Further embodiments of the invention include isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to (a) the nucleotide sequence of an ORF described in Table 2(a) or 3, (b) the nucleotide sequence of an ORF described in Table 2(a) or 3, but lacking the codon for the N-terminal methionine residue, if present, or (c) a nucleotide sequence complementary to any of the nucleotide sequences in (a) or (b). By a polynucleotide having a nucleotide sequence at least, for example, 95% identical to the reference M. jannaschii ORF nucleotide sequence is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the ORF sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference ORF nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

As a practical matter, whether any particular nucleic acid molecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence of a M. jannaschii ORF can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

Preferred are nucleic acid molecules having sequences at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of a M. jannaschii ORF that encode a functional polypeptide. By a “functional polypeptide” is intended a polypeptide exhibiting activity similar, but not necessarily identical, to an activity of the protein encoded by the M. jannaschii ORF. For example, the M. jannaschii ORF MJ1434 encodes an endonuclease that degrades DNA. Thus, a “functional polypeptide” encoded by a nucleic acid molecule having a nucleotide sequence, for example, 95% identical to the nucleotide sequence of MJ1434, will also degrade DNA. As the skilled artisan will appreciate, assays for determining whether a particular polypeptide is “functional” will depend on which ORF is used as the reference sequence. Depending on the reference ORF, the assay chosen for measuring polypeptide activity will be readily apparent in light of the role categories provided in Table 2(a).

Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of a reference ORF will encode a functional polypeptide. In fact, since degenerate variants all encode the same amino acid sequence, this will be clear to the skilled artisan even without performing a comparison assay for protein activity. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a functional polypeptide. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly affect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid).

For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U. et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990), wherein the authors indicate that there are two main approaches for studying the tolerance of an amino acid sequence to change. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selections or screens to identify sequences that maintain functionality. As the authors state, these studies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at a certain position of the protein. For example, most buried amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described in Bowie, J. U. et al., supra, and the references cited therein.

The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated nucleic acid molecule having the nucleotide sequence of a M. jannaschii ORF is intended fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length that are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 50-500 nt in length are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of a M. jannaschii ORF. By a fragment at least 20 nt in length, for example, is intended fragments that include 20 or more contiguous bases from the nucleotide sequence of a M. jannaschii ORF. Since M. jannaschii ORFs are listed in Tables 2(a) and 3 and the genome sequence has been provided, generating such DNA fragments would be routine to the skilled artisan. For example, restriction endonuclease cleavage or shearing by sonication could easily be used to generate fragments of various sizes. Alternatively, such fragments could be generated synthetically.

Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding epitope-bearing portions of a M. jannaschii protein. Methods for determining such epitope-bearing portions are described in detail below.

In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide that hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, an ORF described in Table 2(a) or 3. By “stringent hybridization conditions” is intended overnight incubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

By a polynucleotide that hybridizes to a “portion” of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below.

Of course, polynucleotides hybridizing to a larger portion of the reference polynucleotide (e.g., a M. jannaschii ORF), for instance, a portion 50-500 nt in length, or even to the entire length of the reference polynucleotide, are also useful as probes according to the present invention, as are polynucleotides corresponding to most, if not all, of a M. jannaschii ORF.

By “expression modulating fragment” (EMF), is intended a series of nucleotides that modulate the expression of an operably linked ORF or EMF. A sequence is said to “modulate the expression of an operably linked sequence” when the expression of the sequence is altered by the presence of the EMF. EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements). One class of EMFs are fragments that induce the expression of an operably linked ORF in response to a specific regulatory factor or physiological event. EMF sequences can be identified within the M. jannaschii genome by their proximity to the ORFs described in Tables 2(a), 2(b), and 3. An intergenic segment, or a fragment of the intergenic segment, from about 10 to 200 nucleotides in length, taken 5′ from any one of the ORFs of Tables 2(a), 2(b) or 3 will modulate the expression of an operably linked 3′ ORF in a fashion similar to that found with the naturally linked ORF sequence. As used herein, an “intergenic segment” refers to the fragments of the M. jannaschii genome that are between two ORF(s) herein described. Alternatively, EMFs can be identified using known EMFs as a target sequence or target motif in the computer-based systems of the present invention.

The presence and activity of an EMF can be confirmed using an EMF trap vector. An EMF trap vector contains a cloning site 5′ to a marker sequence. A marker sequence encodes an identifiable phenotype, such as antibiotic resistance or a complementing nutrition auxotrophic factor, which can be identified or assayed when the EMF trap vector is placed within an appropriate host under appropriate conditions. As described above, an EMF will modulate the expression of an operably linked marker sequence. A more detailed discussion of various marker sequences is provided below.

A sequence that is suspected as being an EMF is cloned in all three reading frames in one or more restriction sites upstream from the marker sequence in the EMF trap vector. The vector is then transformed into an appropriate host using known procedures and the phenotype of the transformed host in examined under appropriate conditions. As described above, an EMF will modulate the expression of an operably linked marker sequence.

By “uptake modulating fragment” (UMF), is intended a series of nucleotides that mediate the uptake of a linked DNA fragment into a cell. UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer-based systems described below. The presence and activity of a UMF can be confirmed by attaching the suspected UMF to a marker sequence. The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions and the uptake of the marker sequence is determined. As described above, a UMF will increase the frequency of uptake of a linked marker sequence.

By a “diagnostic fragment” (DF), is intended a series of nucleotides that selectively hybridize to M. jannaschii sequences. DFs can be readily identified by identifying unique sequences within the M. jannaschii genome, or by generating and testing probes or amplification primers consisting of the DF sequence in an appropriate diagnostic format for amplification or hybridization selectivity.

Each of the ORFs of the M. jannaschii genome disclosed in Tables 2(a) and 3, and the EMF found 5′ to the ORF, can be used in numerous ways as polynucleotide reagents. The sequences can be used as diagnostic probes or diagnostic amplification primers to detect the presence M. jannaschii in a sample. This is especially the case with the fragments or ORFs of Table 3, which will be highly selective for M. jannaschii.

In addition, the fragments of the present invention, as broadly described, can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of a polynucleotide sequence to DNA or RNA. Polynucleotides suitable for use in these methods are usually 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).

Triple helix- formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide.

Vectors and Host Cells

The present invention further provides recombinant constructs comprising one or more fragments of the M. jannaschii genome. The recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which, for example, a M. jannaschii ORF is inserted. The vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF. For vectors comprising the EMFs and UMFs of the present invention, the vector may further comprise a marker sequence or heterologous ORF operably linked to the EMF or UMF. Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention. The following vectors are provided by way of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), and trc. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

The present invention further provides host cells containing any one of the isolated fragments (preferably an ORF) of the M. jannaschii genome described herein. The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the can be a procaryotic cell, such as a bacterial cell. Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)). Host cells containing, for example, a M. jannaschii ORF can be used conventionally to produce the encoded protein.

Polypeptides and Fragments

The invention further provides an isolated polypeptide encoded by a M. jannaschii ORF described in Tables 2(a) or 3, or a peptide or polypeptide comprising a portion of the isolated polypeptide. The terms “peptide” and “oligopeptide” are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires to indicate a chain of at least two amino acids coupled by peptidyl linkages. The word “polypeptide” is used herein for chains containing more than ten amino acid residues.

It will be recognized in the art that some amino acid sequence of the M. jannaschii polypeptide can be varied without significant affect of the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity. In general, it is possible to replace residues which form the tertiary structure, provided that residues performing a similar function are used. In other instances, the type of residue may be completely unimportant if the alteration occurs at a non-critical region of the protein.

Thus, the invention further includes variations of a M. jannaschii protein encoded by an ORF described in Table 2(a) or 3 that show substantial protein activity. Methods for assaying such “functional polypeptides” for protein activity are described above. Variations include deletions, insertions, inversions, repeats, and type substitutions (for example, substituting one hydrophilic residue for another, but not strongly hydrophilic for strongly hydrophobic as a rule). Small changes or such “neutral” amino acid substitutions will generally have little effect on protein activity.

Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.

As indicated in detail above, further guidance concerning amino acid changes that are likely to be phenotypically silent (i.e., are not likely to have a significant deleterious effect on function) can be found in Bowie, J. U., et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990).

The fragment, derivative, variant or analog of a M. jannaschii polypeptide encoded by an ORF described in Table 2(a) or 3, may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

Of particular interest are substitutions of charged amino acids with another charged amino acid and with neutral or negatively charged amino acids. The latter results in proteins with reduced positive charge to improve the characteristics of a M. jannaschii ORF-encoded protein. The prevention of aggregation is highly desirable. Aggregation of proteins not only results in a loss of activity but can also be problematic when preparing pharmaceutical formulations, because they can be immunogenic. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993)).

As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 1).

TABLE 1 Conservative Amino Acid Substitutions. Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

Amino acids in a M. jannaschii ORF-encoded protein of the present invention that are essential for function can be identified by methods known in the art, such a s site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutaions at every residue in the molecule.

The polypeptides of the present invention are preferably provided in an isolated form. By “isolated polypeptide” is intended a polypeptide removed from its native environment. Thus, a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention. Also intended as an “isolated polypeptide” are polypeptides that hive been purified, partially or substantially, from a recombinant host cell. For example, a recombinant produced version of a M. jannaschii ORF-encoded protein can be substantially purified by the one-step method described in Smith and Johnson, Gene 67.231-40 (1988).

The polypeptides of the present invention include the proteins encoded by (a) an ORF described in Table 2(a) or 3 or (b) an ORF described in Table 2(a) or 3, but minus the codon for the N-terminal methionine residue, if present, as well as polypeptides that have at least 90% similarity, more preferably at least 95% similarity, and still more preferably at least 96%, 97%, 98% or 99% similarity to a M. jannaschii ORF-encoded protein. Further polypeptides of the present invention include polypeptides at least 90% identical, more preferably at least 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to a M. jannaschii ORF-encoded protein.

By “% similarity” for two polypeptides is intended a similarity score produced by comparing the amino acid sequences of the two polypeptides using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) and the default settings for determining similarity. Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489, 1981) to find the best segment of similarity between two sequences.

By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a reference amino acid sequence of a M. jannaschii ORF-encoded protein is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide has an amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of a M. jannaschii ORF-encoded protein can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.

As described in detail below, the polypeptides of the present invention can also be used to raise polyclonal and monoclonal antibodies, which are useful in assays for detecting M. jannaschii protein expression.

In another aspect, the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention. The epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention. An “immunogenic epitope” is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. These immunogenic epitopes are believed to be confined to a few loci on the molecule. On the other hand, a region of a protein molecule to which an antibody can bind is defined as an “antigenic epitope.” The number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).

As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R. A. (1983). Antibodies that react with predetermined sites on proteins are described in Science 219:660-666. Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals. Peptides that are extremely hydrophobic and those of six or fewer residues generally are ineffective at inducing antibodies that bind to the mimicked protein; longer, peptides, especially those containing proline residues, usually are effective. Sutcliffe et al., supra, at 661. For instance, 18 of 20 peptides designed according to these guidelines, containing 8-39 residues covering 75% of the sequence of the influenza virus hemagglutinin HAI polypeptide chain, induced antibodies that reacted with the HAI protein or intact virus; and 12/12 peptides from the MuLV polymerase and 18/18 from the rabies glycoprotein induced antibodies that precipitated the respective proteins.

Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. Thus, a high proportion of hybridomas obtained by fusion of spleen cells from donors immunized with an antigen epitope-bearing peptide generally secrete antibody reactive with the native protein. Sutcliffe et al., supra, at 663. The antibodies raised by antigenic epitope-bearing peptides or polypeptides are useful to detect the mimicked protein, and antibodies to different peptides may be used for tracking the fate of various regions of a protein precursor which undergoes post-translational processing. The peptides and anti-peptide antibodies may be used in a variety of qualitative or quantitative assays for the mimicked protein, for instance in competition assays since it has been shown that even short peptides (e.g., about 9 amino acids) can bind and displace the larger peptides in immunoprecipitation assays. See, for instance, Wilson et al., Cell 37:767-778 (1984) at 777. The anti-peptide antibodies of the invention also are useful for purification of the mimicked protein, for instance, by adsorption chromatography using methods well known in the art.

Antigenic epitope-bearing peptides and polypeptides of the invention designed according to the above guidelines preferably contain a sequence of at least seven, more preferably at least nine and most preferably between about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention. However, peptides or polypeptides comprising a larger portion of an amino acid sequence of a polypeptide of the invention, containing about 30 to about 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are considered epitope-bearing peptides or polypeptides of the invention and also are useful for inducing antibodies that react with the mimicked protein. Preferably, the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues and highly hydrophobic sequences are preferably avoided); and sequences containing proline residues are particularly preferred.

The epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means for making peptides or polypeptides including recombinant means using nucleic acid molecules of the invention. For instance, a short epitope-bearing amino acid sequence may be fused to a larger polypeptide which acts as a carrier during recombinant production and purification, as well as during immunization to produce anti-peptide antibodies. Epitope-bearing peptides also may be synthesized using known methods of chemical synthesis. For instance, Houghten has described a simple method for synthesis of large numbers of peptides, such as 10-20 mg of 248 different 13 residue peptides representing single amino acid variants of a segment of the HA1 polypeptide which were prepared and characterized (by ELISA-type binding studies) in less than four weeks. Houghten, R. A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA 82:5131-5135. This “Simultaneous Multiple Peptide Synthesis (SMPS)” process is further described in U.S. Pat. No. 4,631,211 to Houghten et al. (1986). In this procedure the individual resins for the solid-phase synthesis of various peptides are contained in separate solvent-permeable packets, enabling the optimal use of the many identical repetitive steps involved in solid-phase methods. A completely manual procedure allows 500-1000 or more syntheses to be conducted simultaneously. Houghten et al, supra, at 5134.

Epitope-bearing peptides and polypeptides of the invention are used to induce antibodies according to methods well known in the art. See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. et al., J. Gen Virol. 66:2347-2354 (1985). Generally, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine may be coupled to carrier using a linker such as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carrier using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 g peptide or carrier protein and Freund's adjuvant. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.

Immunogenic epitope-bearing peptides of the invention, i.e., those parts of a protein that elicit an antibody response when the whole protein is the immunogen, are identified according to methods known in the art. For instance, Geysen et al., supra, discloses a procedure for rapid concurrent synthesis on solid supports of hundreds of peptides of sufficient purity to react in an enzyme-linked immunosorbent assay. Interaction of synthesized peptides with antibodies is then easily detected without removing them from the support. In this manner a peptide bearing an immunogenic epitope of a desired protein may be identified routinely by one of ordinary skill in the art For instance, the immunologically important epitope in the coat protein of foot-and-mouth disease virus was located by Geysen et al. with a resolution of seven amino acids by synthesis of an overlapping set of all 208 possible hexapeptides covering the entire 213 amino acid sequence of the protein. Then, a complete replacement set of peptides in which all 20 amino acids were substituted in turn at every position within the epitope were synthesized, and the particular amino acids conferring specificity for the reaction with antibody were determined. Thus, peptide analogs of the epitope-bearing peptides of the invention can be made routinely by this method. U.S. Pat. No. 4,708,781 to Geysen (1987) further describes this method of identifying a peptide bearing an immunogenic epitope of a desired protein.

Further still, U.S. Pat. No. 5,194,392 to Geysen (1990) describes a general method of detecting or determining the sequence of monomers (amino acids or other compounds) which is a topological equivalent of the epitope (i.e., a “mimotope”) which is complementary to a particular paratope (antigen binding site) of an antibody of interest. More generally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a method of detecting or determining a sequence of monomer which is a topographical equivalent of a ligand which is complementary to the ligand binding site of a particular receptor of interest. Similarly, U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996) on Peralkylated Oligopeptide Mixtures discloses linear C₁-C₇-alkyl peralkylated oligopeptides and sets and libraries of such peptides, as well as methods for using such oligopeptide sets and libraries for determining the sequence of a peralkylated oligopeptide that preferentially binds to an acceptor molecule of interest. Thus, non-peptide analogs of the epitope-bearing peptides of the invention also can be made routinely by these methods.

The entire disclosure of each document cited in this section on “Polypeptides and Peptides” is hereby incorporated herein by reference.

As one of skill in the art will appreciate, the polypeptides of the present invention and the epitope-bearing fragments thereof described above can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. This has been demonstrated, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84-86 (1988)). Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric protein or protein fragment alone (Fountoulakis et al., J Biochem 270:3958-3964 (1995)).

Protein Function

Each ORF described in Table 2(a) was assigned to biological role categories adapted from Riley, M., Microbiology Reviews 57(4):862 (1993)). This allows the skilled artisan to determine a function for each identified coding sequence. For example, a partial list of the M. jannaschii protein functions provided in Table 2(a) includes: methanogenesis, amino acid biosynthesis, cell division, detoxification, protein secretion, transformation, central intermediary metabolism, energy metabolism, degradation of DNA, DNA replication, restriction, modification, recombination and repair, transcription, RNA processing, translation, degradation of proteins, peptides and glycopeptides, ribosomal proteins, translation factors, transport, tRNA modification, and drug and analog sensitivity. A more detailed description of several of these functions is provided in Example 1 below.

Diagnostic Assays

The present invention further provides methods to identify the expression of an ORF of the present invention, or homolog thereof, in a test sample, using one of the DFs or antibodies of the present invention. Such methods involve incubating a test sample with one or more of the antibodies or one or more of the DFs of the present invention and assaying for binding of the DFs or antibodies to components within the test sample.

Conditions for incubating a DF or antibody with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the DF or antibody used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the DFs or antibodies of the present invention. Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein or membrane extracts of cells. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is compatible with the system utilized.

In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention. Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers including comprising: (a) a first container comprising one of the DFs or antibodies of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound DF or antibody.

A compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allow one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the antibodies used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound antibody or DF.

Types of detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents that are capable of reacting with the labeled antibody. One skilled in the art will readily recognize that the disclosed DFs and antibodies of the present invention can be readily incorporated into one of the established kit formats that are well known in the art.

Screening Assay for Binding Agents

Using the isolated proteins described herein, the present invention further provides methods of obtaining and identifying agents that bind to a protein encoded by a M. jannaschii ORF or to a fragment thereof.

The method involves:

(a) contacting an agent with an isolated protein encoded by a M. jannaschii ORF, or an isolated fragment thereof; and

(b) determining whether the agent binds to said protein or said fragment.

The agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents. The agents can be selected and screened at random or rationally selected or designed using protein modeling techniques. For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to the protein encoded by an ORF of the present invention.

Alternatively, agents may be rationally selected or designed. As used herein, an agent is said to be “rationally selected or designed” when the agent is chosen based on the configuration of the particular protein. For example, one skilled in the art can readily adapt currently available procedures to generate peptides, pharmaceutical agents and the like capable of binding to a specific peptide sequence in order to generate rationally designed antipeptide peptides, for example see Hurby et al., Application of Synthetic Peptides: Antisense Peptides, In Synthetic Peptides, A User's Guide, W. H. Freeman, NY (1992), pp. 289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or the like.

In addition to the foregoing, one class of agents of the present invention, can be used to control gene expression through binding to one of the ORFs or EMFs of the present invention. As described above, such agents can be randomly screened or rationally designed and selected. Targeting the ORF or EMF allows a skilled artisan to design sequence specific or element specific agents, modulating the expression of either a single ORF or multiple ORFs that rely on the same EMF for expression control.

One class of DNA binding agents are those that contain nucleotide base residues that hybridize or form a triple helix by binding to DNA or RNA. Such agents can be based on the classic phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl or polymeric derivatives having base attachment capacity.

Agents suitable for use in these methods usually contain 20 to 40 bases and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al, Science 241:456 (1988); and Dervan et al., Science 251: 1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide and other DNA binding agents.

Computer Related Embodiments

The nucleotide sequence provided in SEQ ID NO:1, 2, or 3, a representative fragment thereof, or a nucleotide sequence at least 99.9% identical to the sequence provided in SEQ ID NO:1, 2, or 3, can be “provided” in a variety of mediums to facilitate use thereof. As used herein, provided refers to a manufacture, other than an isolated nucleic acid molecule, that contains a nucleotide sequence of the present invention, i.e., the nucleotide sequence provided in SEQ ID NO:1, 2, or 3, a representative fragment thereof, or a nucleotide sequence at least 99.9% identical to SEQ ID NO:1, 2, or 3. Such a manufacture provides the M. jannaschii genome or a subset thereof (e.g., a M. jannaschii open reading frame (ORF)) in a form that allows a skilled artisan to examine the manufacture using means not directly applicable to examining the M. jannaschii genome or a subset thereof as it exists in nature or in purified form.

In one application of this embodiment, a nucleotide sequence of the present invention can be recorded on computer readable media. As used herein, “computer readable media” refers to any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. A skilled artisan can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide sequence of the present invention.

As used herein, “recorded” refers to a process for storing information on computer readable medium. A skilled artisan can readily adopt any of the presently know methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention. A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and MicroSoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. A skilled artisan can readily adapt any number of dataprocessor structuring formats (e.g. text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.

By providing the nucleotide sequence of SEQ ID NO:1, 2, or 3, a representative fragment thereof, or a nucleotide sequence at least 99.9% identical to SEQ ID NO:1, 2, or 3, in computer readable form, a skilled artisan can routinely access the sequence information for a variety of purposes. Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium. The examples which follow demonstrate how software which implements the BLAST (Altschul et al., J. Mol. Biol. 215:403410 (1990)) and BLAZE (Brutiag et al., Comp. Chem. 17:203-207 (1993)) search algorithms on a Sybase system can be used to identify open reading frames (ORFs) within the M. jannaschii genome that contain homology to ORFs or proteins from other organisms. Such ORFs are protein-encoding fragments within the M. jannaschii genome and are useful in producing commercially important proteins such as enzymes used in methanogenesis, amino acid biosynthesis, metabolism, fermentation, transcription, translation, RNA processing, nucleic acid and protein degradation, protein modification, and DNA replication, restriction, modification, recombination, and repair. A comprehensive list of ORFs encoding commercially important M. jannaschii proteins is provided in Tables 2(a) and 3.

The present invention further provides systems, particularly computer-based systems, which contain the sequence information described herein. Such systems are designed to identify commercially important fragments of the M. jannaschii genome. As used herein, “a computer-based system” refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention. The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means. A skilled artisan can readily appreciate that any one of the currently available computer-based system are suitable for use in the present invention.

As indicated above, the computer-based systems of the present invention comprise a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means. As used herein, “data storage means” refers to memory that can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention. As used herein, “search means” refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of the M. jannaschii genome that match a particular target sequence or target motif. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are available and can be used in the computer-based systems of the present invention. Examples of such software include, but are not limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA). A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting homology searches can be adapted for use in the present computer-based systems.

As used herein, a “target sequence” can be any DNA or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. The most preferred sequence length of a target sequence is from about 10 to 100 amino acids or from about 30 to 300 nucleotide residues. However, it is well recognized that during searches for commercially important fragments of the M. jannaschii genome, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.

As used herein, “a target structural motif,” or “target motif,” refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif. There are a variety of target motifs known in the art. Protein target motifs include, but are not limited to, enzymic active sites and signal sequences. Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).

Thus, the present invention further provides an input means for receiving a target sequence, a data storage means for storing the target sequence and the homologous M. jannaschii sequence identified using a search means as described above, and an output means for outputting the identified homologous M. jannaschii sequence. A variety of structural formats for the input and output means can be used to input and output information in the computer-based systems of the present invention. A preferred format for an output means ranks fragments of the M. jannaschii genome possessing varying degrees of homology to the target sequence or target motif. Such presentation provides a skilled artisan with a ranking of sequences which contain various amounts of the target sequence or target motif and identifies the degree of homology contained in the identified fragment.

A variety of comparing means can be used to compare a target sequence or target motif with the data storage means to identify sequence fragments of the M. jannaschii genome. For example, implementing software which implement the BLAST and BLAZE algorithms (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) can be used to identify open reading frames within the M. jannaschii genome. A skilled artisan can readily recognize that any one of the publicly available homology search programs can be used as the search means for the computer-based systems of the present invention.

One application of this embodiment is provided in FIG. 8. FIG. 8 provides a block diagram of a computer system 102 that can be used to implement the present invention. The computer system 102 includes a processor 106 connected to a bus 104. Also connected to the bus 104 are a main memory 108 (preferably implemented as random access memory, RAM) and a variety of secondary storage devices 110, such as a hard drive 112 and a removable medium storage device 114. The removable medium storage device 114 may represent, for example, a floppy disk drive, a CD-ROM drive, a magnetic tape drive, etc. A removable storage medium 116 (such as a floppy disk, a compact disk, a magnetic tape, etc.) containing control logic and/or data recorded therein may be inserted into the removable medium storage device 114. The computer system 102 includes appropriate software for reading the control logic and/or the data from the removable medium storage device 114 once inserted in the removable medium storage device 114.

A nucleotide sequence of the present invention may be stored in a well known manner in the main memory 108, any of the secondary storage devices 110, and/or a removable storage medium 116. Software for accessing and processing the genomic sequence (such as search tools, comparing tools, etc.) reside in main memory 108 during execution.

Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.

EXPERIMENTAL COMPLETE GENOME SEQUENCE OF THE METHANOGENIC ARCHAEON, METHANOCOCCUS JANNASCHII EXAMPLE 1

A whole genome random sequencing method (Fleischmann, R. D., et al., Science 269:496 (1995); Fraser, C. M., et al., Science 270:397 (1995)) was used to obtain the complete genome sequence for M. jannaschii. A small insert plasmid library (2.5 Kbp average insert size) and a large insert lambda library (16 Kbp average insert size) were used as substrates for sequencing. The lambda library was used to form a genome scaffold and to verify the orientation and integrity of the contigs formed from the assembly of sequences from the plasmid library. All clones were sequenced from both ends to aid in ordering of contigs during the sequence assembly process. The average length of sequencing reads was 481 bp. A total of 36,718 sequences were assembled by means of the TIGR Assembler (Fleischmann, R. D., et al., Science 269:496 (1995); Fraser, C. M., et al., Science 270:397 (1995); Sutton G., et al, Genome Sci. Tech. 1:9 (1995)). Sequence and physical gaps were closed using a combination of strategies (Fleischmann, R. D., et al., Science 269:496 (1995); Fraser, C. M., et al., Science 270:397 (1995)). The colinearity of the in vivo genome to the genome sequence was confirmed by comparing restriction fragments from six, rare cutter, restriction enzymes (Aat II, BamHI, Bgl II, Kpn I, Sma I, and Sst II) to those predicted from the sequence data Additional confidence in the colinearity was provided by the genome scaffold produced by sequence pairs from 339 large-insert lambda clones, which covered 88% of the main chromosome. Open reading frames (ORFs) and predicted protein-coding regions were identified as described (Fleischmann, R. D., et al., Science 269:496 (1995); Fraser, C. M., et al., Science 270:397 (1995)) with some modification. In particular, the statistical prediction of M. jannaschii genes was performed with GeneMark (Borodovsky, M. & McIninch, J. Comput. Chem. 17:123 (1993)). Regular GeneMark uses nonhomogeneous Markov models derived from a training set of coding sequences and ordinary Markov models derived from a training set of noncoding sequences. Only a single 16S ribosomal RNA sequence of M. jannaschii was available in the public sequence databases before the whole genome sequence described here. Thus, the initial training set to determine parameters of a coding sequence Markov model was chosen as a set of ORFs>1000 nucleotides (nt). As an initial model for non-coding sequences, a zero-order Markov model with genome-specific nucleotide frequencies was used. The initial models were used at the first prediction step. The results of the first prediction were then used to compile a set of putative genes used at the second training step. Alternate rounds of training and predicting were continued until the set of predicted genes stabilized and the parameters of the final fourth-order model of coding sequences were derived. The regions predicted as noncoding were then used as a training set for a final model for noncoding regions. Cross-validaton simulations demonstrated that the GeneMark program trained as described above was able to correctly identify coding regions of at least 96 nt in 94% of the cases and noncoding regions of the same length in 96% of the cases. These values assume that the self-training method produced correct sequence annotation for compiled control sets. Comparison with the results obtained by searches against a nonredundant protein database (Fleischmann, R. D., et al., Science 269:496 (1995); Fraser, C. M., et al., Science 270:397 (1995)) demonstrated that almost all genes identified by sequence similarity were predicted by the GeneMark program as well. This observation provides additional confidence in genes predicted by GeneMark whose protein translations did not show significant similarity to known protein sequences. The predicted protein-coding regions were search against the Blocks database (Henikoff, S. & Henikoff, J. G., Genomics 19:97 (1994)] by means of BLIMPS (Wallace, J. C. & Henikoff, S., CABIOS 8:249 (1992)) to verify putative identifications and to identify potential functional motifs in predicted protein-coding regions that had no database match. Genes were assigned to known metabolic pathways. When a gene appeared to be missing from a pathway, the unassigned ORFs and the complete M. jannaschii genome sequence were searched with specific query sequences or motifs from the Blocks database. Hydrophobicity plots were performed on all predicted protein-coding regions by means of the Kyte-Doolittle algorithm (Kyte, J. & Doolittle, R. F., J. Mol. Biol. 157:105 (1982)) to identify potentially functionally relevant signatures in these sequences.

The M. jannaschii genome comprises three physically distinct elements: i) a large circular chromosome of 1,664,976 base pairs (bp) (SEQ ID NO:1), which contains 1682 predicted protein-coding regions and has a G+C content of 31.4%; ii) a large circular extrachromosomal element (ECE) (Zhao, H., et al., Arch. Microbiol. 150:178 (1988)) of 58,407 bp (SEQ ID NO:2), which contains 44 predicted protein coding regions and has a G+C content of 28.2% (FIG. 3); and iii) a small circular ECE (Zhao, H., et al., Arch. Microbiol. 150:178 (1988)) of 16,550 bp (SEQ ID NO:3), which contains 12 predicted protein coding regions, and has a G+C content of 28.8% (FIG. 3). With respect to its shape, size, G+C content, and gene density the main chromosome resembles that of H. influenzae. However, here the resemblance stops.

Of the 1743 predicted protein-coding regions reported previously for H. influenzae, 78% had a match in the public sequence database (Fleischmann, R. D., et al., Science 269:496 (1995); Fraser, C. M., et al, Science 270:397(1995)). Of these, 58% were matches to genes with reasonably well defined function, while 20% were matches to genes whose function was undefined. Similar observations were made for the M. genitalium genome (Fleischmann, R. D., et al., Science 269:496 (1995); Fraser, C. M., et al., Science 270:397 (1995)). Eighty-three percent of the predicted protein coding regions from M. genitalium have a counterpart in the H. influenzae genome. In contrast, only 38% of the predicted protein-coding regions from M. jannaschii match a gene in the database that could be assigned a putative cellular role with high confidence; 6% of the predicted protein-coding regions had matches to hypothetical proteins (FIG. 4; Tables 2-3). Approximately 100 genes in M. jannaschii had marginal similarity to genes or segments of genes from the public sequence databases and could not be assigned a putative cellular role with high confidence. Only 11% of the predicted protein-coding regions from H. influenzae and 17% of the predicted protein coding regions from M. genitalium matched a predicted protein coding region from M. jannaschii. Clearly the M. jannaschii genome, and undoubtedly, therefore, all archaeal genomes are remarkably unique, as the phylogenetic position of these organisms would suggest.

Energy production in M. jannaschii occurs via the reduction of CO₂ with H₂ to produce methane. Genes for all of the known enzymes and enzyme complexes associated with methanogenesis (DiMarco, A. A., et al., Ann. Rev. Biochem. 59:355 (1990)) were identified in M. jannaschii, the sequence and order of which are typical of methanogens. M. jannaschii appears to use both H₂ and formate as substrates for methanogenesis, but lacks the genes to use methanol or acetate. The ability to fix nitrogen has been demonstrated in a number of methanogens (Belay, N., et al., Nature 312:286 (1984)) and all of the genes necessary for this pathway have been identified in M. jannaschii (Tables 2-3). In addition to its anabolic pathways, several scavenging molecules have been identified in M. jannaschii that probably play a role in importing small organic compounds, such as amino acids, from the environment (Tables 2-3).

Three different pathways are known for the fixation of CO₂ into organic carbon: the non-cyclic, reductive acetyl-coenzyme A-carbon monoxide dehydrogenase pathway (Ljungdahl-Wood pathway), the reductive trichloroacetic acid (TCA) cycle, and the Calvin cycle. Methanogens fix carbon by the Ljungdahl-Wood pathway (Wood, H. G., et al., TIBS 11:14 (1986)), which is facilitated by the carbon monoxide dehydrogenease enzyme complex (CODH) (Blaat, M., Antonie van Leewenhoek 66:187 (1994)). The complete Ljungdahl-Wood pathway, encoded in the M. jannaschii genome, depends on the methyl carbon in methanogenesis; however, methanogenesis can occur independently of carbon fixation.

Although genes encoding two enzymes required for gluconeogenesis (glucopyruvate oxidoreductase and phosphoenolpyruvate synthase) were found in the M. jannaschii genome, genes encoding other key intermediates of gluconeogenesis (fructose bisphosphatase and fructose 1,6-bisphosphate aldolase) were not been identified. Glucose catabolism by glycolysis also requires the aldolase, as well as phosphofructokinase, an enzyme that also was not found in M. jannaschii and has not been detected in any of the Archaea. In addition, genes specific for the Entner-Doudoroff pathway, an alternative pathway used by some microbes for the catabolism of glucose, were not identified in the genomic sequence. The presence of a number of nearly complete metabolic pathways suggests that some key genes are not recognizable at the sequence level, although we cannot exclude the possibility that M. jannaschii may use alternative metabolic pathways.

In general, M. jannaschii genes that encode proteins involved in the transport of small inorganic ions into the cell are homologs of bacterial genes. The genome includes many representatives of the ABC transporter family, as well as genes for exporting heavy metals (e.g., the chromate-resistance protein) and other toxic compounds (e.g the nor A drug efflux pump locus).

More than 20 predicted protein-coding regions have sequence similarity to polysaccharide biosynthetic enzymes. These genes have only bacterial homologs or are most closely related to their bacterial counterparts. The identified polysaccharide biosynthetic genes in M. jannaschii include those for the interconversion of sugars, activation of sugars to nucleotide sugars, and glycosyltransferases for the polymerization of nucleotide sugars into oligo- and polysaccharides that are subsequently incorporated into surface structures (Hartmann, E. and König, H., Arch Microbiol. 151:274 (1989)). In an arrangement reminiscent of bacterial polysaccharide biosynthesis genes, many of the genes for M. jannaschii polysaccharide production are clustered together (Tables 2-3, FIG. 4). The G+C content in this region is <95% of that in the rest of the M. jannaschii genome. A similar observation was made in Salmonella typhimurium (Jiang, X. M., et al., Mol. Microbiol. 5:695 (1991)) in which the gene cluster for lipopolysaccharide 0 antigen has a significantly lower G+C ratio than the rest of the genome. In that case, the difference in G+C content was interpreted as meaning that the region originated by lateral transfer from another organism.

Of the three main multicomponent information processing systems (transcription, translation, and replication), translation appears the most universal in its overall makeup in that the basic translation machinery is similar in all three domains of life. M. jannaschii has two ribosomal RNA operons, designated A and B, and a separate 5S RNA gene that is associated with several transfer RNAs (tRNAs). Operon A has the organization, 16S—23S—5S, whereas operon B lacks the 5S component An alanine tRNA is situated in the spacer region between the 16S and 23S subunits in both operons. The majority of proteins associated with the ribosomal subunits (especially the small subunit) are present in both Bacteria and Eukaryotes. However, the relatively protein-rich eukaryotic ribosome contains additional ribosomal proteins not found in the bacterial ribosome. A smaller number of bacteria-specific ribosomal proteins exist as well. The M. jannaschii genome contains all ribosomal proteins that are common to eukaryotes and bacteria. It shows no homologs of the bacterial-specific ribosomal proteins, but does possess homologs of a number of the eukaryotic-specific ones. Homologs of all archaea-specific ribosomal proteins that have been reported to date (Lechner, K., et al., J. Mol. Evol. 29:20 (1989); Köpke, A. K. E. and Wittimann-Liebold, B., Can. J. Microbiol. 35:11 (1989)) are found in M. jannaschii.

As previously shown for other archaea (Iwabe, N., et al., Proc. Natl. Acad. Sci. USA 86:9355 (1989); Gogarten J. P., et al., Proc. Natl. Acad. Sci. USA 86:6661 (1989); Brown, J. R. and Doolittle, W. F., Proc. Natl. Acad. Sci. USA 92:2441 (1995)), the Methanococcus translation elongation factors EF-1α (EF-Tu in bacteria) and EF-2 (EF-G in bacteria) are most similar to their eukaryotic counterparts. In addition, the M. jannaschii genome contains 11 translation initiation factor genes. Three of these genes encode the subunits homologous to those of the eukaryotic IF-2, and are reported here in the Archaea for the first time. A fourth initiation factor gene that encodes a second IF-2 is also found in M. jannaschii. This additional IF-2 gene is most closely related to the yeast protein FUN12 which, in turn, appears to be a homolog of the bacterial IF-2. It is not known which of the two IF-2-like initiation factors identified in M. jannaschii plays a role in directing the initiator tRNA to the start site of the mRNA. The fifth identified initiation factor gene in M. jannaschii encodes IF-1A, which has no bacterial homolog. The sixth gene encodes the hypusine-containing initiation factor eIF-5a. Two subunits of the translation initiation factor eIF-2B were identified in M. jannaschii. Finally, three putative adenososine 5′-triphosphate (ATP)-dependent helicases were identified that belong to the eIF-4a family of translation initiation factors.

Thirty-seven tRNA genes were identified in the M. jannaschii genome. Almost all amino acids encoded by two codons have a single TRNA, except for glutamic acid, which has two. Both an initiator and an internal methionyl tRNA are present. The two pyrimidine-ending isoleucine codons are covered by a single tRNA, while the third (AUA) seems covered by a related tRNA having a CAU anticodon. A single tRNA appears to cover the three isoleucine codons. Those amino acids encoded by f our codons each have two tRNAs, one to cover the Y-, the other the R-ending, codons. Valine has a third tRNA, which is specific for the GUG codon; and alanine has three tRNAs (two of which are in the spacer regions separating the 16S and 23S subunits in the two ribosomal RNA operons). Leucine, serine and arginine, all of which have six codons, each posses three corresponding tRNAs. The genes for the internal methionine and tryptophan tRNAs contain introns in the region of their anti-codon loops.

A tRNA also exists for selenocysteine UGA codon). At least four genes in M. jannaschii contain internal stop codons that are potential selenocysteine codons: the α chain of formate dehydrogenase, coenzyme F420 reducin hydrognase, β-chain tungsten formyl methanofuran dehydrogenase, and a heterodisulfide reductase. Three genes with a putative role in selenocysteine metabolism were identified by their similarity to the sel genes from other organisms (Tables 2-3).

Recognizable homologs for four of the aminoacyl-tRNA synthetases (glutamine, asparagine, lysine, and cysteine) were not identified in the M. jannaschii genome. The absence of a glutaminyl-tRNA synthetase is not surprising in that a number of organisms, including at least one archaeon, have none (Wilcox, M., Eur. J Biochem. 11:405 (1969); Martin, N. C., et al., J. Mol. Biol. 101:285 (1976); Martin, N. C., et al., Biochemistry 16:4672 (1977); Schon, A., et al., Biochimie 70:391 (1988); Soll, D. and RajBhandary, U., Eds. Am. Soc. for Microbiol. (1995)). In these instances, glutaminyl tRNA charging involves a post-charging conversion mechanism whereby the tRNA is charged by the glutamyl-tRNA synthetase with glutamic acid, which then is enzymatically converted to glutamrine. A post-charging conversion is also involved in selenocysteine charging via the seryl-tRNA synthetase. A similar mechanism has been proposed for asparagine charging, but has never been demonstrated (Wilcox, M., Eur. J. Biochem. 11:405 (1969); Martin, N. C., et al., J. Mol. Biol. 101:285 (1976); Martin, N. C., et al., Biochemistry 16:4672 (1977); Schon, A., et al., Biochimie 70:391 (1988); Soll, D. and RajBhandary, U., Eds. Am. Soc. for Microbiol. (1995)). The inability to find homologs of the lysine and cysteine aminoacyl-tRNA synthetases is surprising because bacterial and eukaryotic versions in each instance show clear homology.

Aminoacyl-tRNA synthetases of M. jannaschii and other archaea resemble eukaryoticsynthetases more closely than they resemble bacterial forms. The tryptophanyl synthetase is one of the more notable examples, because the M. jannaschii and eukaryotic version do not appear to be specifically related to the bacterial version (de Pouplana, R., et al., Proc. Natl. Acad. Sci., USA 93:166 (1996)). Two versions of the glycyl synthetase are known in bacteria, one that is very unlike the version found in Archaea and Eukaryote and one that is an obvious homolog of it (Wagner, E. A., et al., J. Bacteriol. 177:5179 (1995); Logan, D. T., et al., EMBO J. 14:4156 (1995)).

Eleven genes encoding subunits of the DNA-dependent RNA polymerase were identified in the M. jannaschii genome. The sequence similarity between the subunits and their homologs in Sutfolobus acidocaldarius supports the evolutionary unity of the archaeal polymerase complex (Woese, C. R. and Wolfe, R. S., Eds. The Bacteria, vol. VIII (Academic Press, NY, 1985); Langer, D., et al., Proc. Natl. Acad. Sci. 92:5768 (1995); Lanzendoerfer, M. et al., System. Appl. Microbiol. 16:656 (1994)). All of the subunits found in M. jannaschii show greater similarity to their eukaryotic counterparts than to the bacterial homologs. The genes encoding the five largest subunits (A′, A″, B′, B″, D) have homologs in all organisms. Six genes encode subunits shared only by Archaea and Eukaryotes (E, H, K, L, and N). The M. jannaschii homolog of the S. acidocaldarius subunit E is split into two genes designated E′ and E″. Sulfolobus acidocaldarius also contains two additional small subunits of RNA polymerase, designated G and F, that have no counterparts in either Bacteria or Eukaryotes. No homolog of these subunits was identified in M. jannaschii.

The archaeal transciption initiation system is essentially the same as that found in Eukaryotes, and is radically different from the bacterial version (Klenk, H. P. and Doolittle, W. F., Curr. Biol. 4.920 (1994)). The central molecules in the former systems are the TATA-binding protein (TBP) and transcription factor B (TFIIB and TFIIIB in Eukaryotes, or simply TFB). In the eukaryotic systems, TBP and TFB are parts of larger complexes, and additional factors (such as TFIIA and TFIIF) are used in the transcription process. However, the M. jannaschii genome does not contain obvious homologs of TFIIA and TFIIF.

Several components of the replication machinery were identified in M. jannaschii. The M. jannaschii genome appears to encode a single DNA-dependent polymerase that is a member of the B family of polymerases (Bernard, A., et al., EMBO J. 6:4219 (1987); Cullman, G., et al., Molec. Cell Biol. 15:4661 (1995); Uemori, T., et al., J. Bacteriol. 117:2164 (1995); Delarue, M., et al., Prot. Engineer. 3:461 (1990); Gavin, K. A., et al., Science 270:1667 (1995)). The polymerase shares sequence similarity and three motifs with other family B polymerases, including eukaryotic α, γ, and ε polymerases, bacterial polymerase II, and several archaeal polymerases. However, it is not homologous to bacterial polymerase I and has no homologs in H. influenzae or M. genitalium.

Primer recognition by the polymerase takes place through a structure-specific DNA binding complex, the replication factor complex (rfc) (Bernard, A., et al., EMBO J. 6:4219 (1987); Cullman, G., et al., Molec. Cell Biol. 15:4661 (1995); Uemori, T., et al., J. Bacteriol. 117:2164 (1995); Delarue, M., et al., Prot. Engineer. 3:461 (1990); Gavin, K. A., et al., Science 270:1667 (1995)). In humans and yeast, the rfc is composed of five proteins: a large subunit and four small subunits that have an associated adenosine triphosphatase (ATPase) activity stimulated by proliferating cell nuclear antigen (PCNA). Two genes in M. jannaschii are putative members of a eukaryotic-like replication factor complex. One of the genes in M. jannaschii is a putative homolog of the large subunit of the rfc, whereas the second is a putative homolog of one of the small subunits. Among Eukaryotes, the rfc proteins share sequence similarity in eight signature domains (Bernard, A., et al., EMBO J. 6:4219 (1987); Cullman, G., et al., Molec. Cell Biol. 15:4661 (1995); Uemori, T., et al., J. Bacteriol. 117:2164 (1995); Delarue, M., et al., Prot. Engineer. 3:461 (1990); Gavin, K. A., et al., Science 270:1667 (1995)). Domain I is conserved only in the large subunit among Eukaryotes and is similar in sequence to DNA ligases. This domain is missing in the large-subunit homolog in M. jannaschii. The remaining domains in the two M. jannaschii genes are well-conserved relative to the eukaryotic homologs. Two features of the sequence similarity in these domains are of particular interest. First, domain II (an ATPase domain) of the small-subunit homolog is split between two highly conserved amino acids (lysine and threonine) by an intervening sequence of unknown function. Second, the sequence of domain VI has regions that are useful for distinguishing between bacterial and eukaryotic rfc proteins (Bernard, A., et al., EMBO J. 6:4219 (1987); Cullman, G., et al., Molec. Cell Biol. 15:4661 (1995); Uemori, T., et al., J. Bacteriol. 117:2164 (1995); Delarue, M., et al., Prot. Engineer. 3:461 (1990); Gavin, K. A., et al., Science 270:1667 (1995)); the rfc sequence for M. jannaschii shares the characteristic eukaryotic signature in this domain.

We have attempted to identify an origin of replication by searching the M. jannaschii genome sequence with a variety of bacterial and eukaryotic replication-origin consensus sequences. Searches with oriC, Co1E1, and autonomously replicating sequences from yeast (Bernard, A., et al., EMBO J. 6:4219 (1987); Cullman, G., et al., Molec. Cell Biol. 15:4661 (1995); Uemori, T., et al., J. Bacteriol. 117:2164 (1995); Delarue, M., et al., Prot. Engineer. 3:461 (1990); Gavin, K. A., et al., Science 270:1667 (1995)) did not identify an origin of replication. With respect to the related cellular processes of replication initiation and cell division, the M. jannaschii genome contains two genes that are putative homologs of Cdc54, a yeast protein that belongs to a family of putative DNA replication initiation proteins (Whitbred, L. A. and Dalton, S., Gene 155:113 (1995)). A third potential regulator of cell division in M. jannaschii is 55% similar at the amino acid level to pelota, a Drosophila protein involved in the regulation of the early phases of meiotic and mitotic cell division (Eberhart, C. G. and Wasserman, S. A., Development 121:3477 (1995)).

In contrast to the putative rfc complex and the initiation of DNA replication, the cell division proteins from M. jannaschii most resemble their bactiial counterparts (Rothfield, L. I. and Zhao, C. R., Cell 84:183 (1996); Lutkenhaus, J., Curr. Opp. Gen. Devel. 3:783 (1993)). Two genes similar to that encoding FtsZ, a ubiquitous bacterial protein, are found in M. jannaschii. FtsZ is a polymer-forming, guanosine triphosphate (GTP)-hydrolyzing protein with tubulin-like elements; it is localized to the site of septation and forms a constricting ring between the dividing cells. One gene similar to FtsJ, a bacterial cell division protein of undetermined function, also is found in M. jannaschii. Three additional genes (MinC, MinD, and MinE) function in concert in Bacteria to determine the site of septation during cell division. In M. jannaschii, three MinD-like genes were identified, but none for MinC or MinE. Neither spindle-associated proteins characteristic of eukaryotic cell division nor bacterial mechanocherical enzymes necessary for partitioning the condensed chromosomes were detected in the M. jannaschii genome. Taken together, these observations raise the possibility that cell division in M. jannaschii might occur via a mechanism specific for the Archaea.

The structural and functional conservation of the signal peptide of secreted proteins in Archaea, Bacteria, and Eukaryotes suggests that the basic mechanisms of membrane targeting and translocation may be similar among all three domains of life. The secretory machinery of M. jannaschii appears a rudimentary apparatus relative to that of bacterial and eukaryotic systems and consists of (i) a signal peptidase (SP) that cleaves the signal peptide of translocating proteins, (ii) a preprotein translocase that is the major constituent of the membrane-localized translocation channel, (iii) a ribonucleoprotein complex (signal recognition particle, SRP) that binds to the signal peptide and guides nascent proteins to the cell membrane, and (iv) a docking protein that acts as a receptor for the SRP. The 7S RNA component of the SRP from M. jannaschii shows a highly conserved structural domain shared by other Archaea, Bacteria, and Eukaryotes (Kaine, B. P. and Merkel, V. L., J. Bacteriol. 171:4261 (1989); Poritz, M. A. et al., Cell 55:4 (1988)). However, the predicted secondary structure of the 7S RNA SRP component in Archaea is more like that found in Eukaryotes than in Bacteria (Kaine, B. P. and Merkel, V. L., J. Bacteriol. 171:4261 (1989); Poritz, M. A. et al., Cell 55:4 (1988)). The SP and docking proteins from M. jannaschii are most similar to their eukaryotic counterparts; the translocase is most similar to the SecY translocation-associated protein in Escherichia coli.

A second distinct signal peptide is found in the flagellin genes of M. jannaschii. Alignment of flagellin genes from M. voltae (Faguy, D. M., et al., Can. J. Microbiol. 40:67 (1994); Kalmokoff, M. L., et al., Arch. Microbiol. 157:481 (1992)) and M. jannaschii reveals a highly conserved NAH₂-terminus (31 of the first 50 residues are identical in all of the mature flagellins). The peptide sequence of the M. jannaschii flagellin indicates that the protein is cleaved after the canonical Gly-12 position, and it is proposed to be similar to type-IV pilins of Bacteria (Faguy, D. M., et al., Can. J. Microbiol. 40:67 (1994); Kalmokoff, M. L., et al., Arch. Microbiol. 157:481 (1992)).

Five histone genes are present in the M. jannaschii genome—three on the main chromosome and two on the large ECE. These genes are homologs of eukaryotic histones (H2a, H2b, H3, and H4) and of the eukaryotic transcription-related CAAT-binding factor CBF-A (Sandman, K., et al., Proc. Natl. Acad. Sci. USA 87:5788 (1990)). The similarity between archaeal and eukaryotic histones suggests that the two groups of organisms resemble one another in the roles histones play both in genome supercoiling dynamics and in gene expression. The five M. jannaschii histone genes show greatest similarity among themselves even though a histone sequence is available from the closely related species, Methanococcus voltae. This intraspecific similarity suggests that the gene duplications that produced the five histone genes occurred on the M. jannaschii lineage per se.

Self-splicing portions of a peptide sequence that generally encode a DNA endonuclease activity are called inteins, in analogy to introns (Kane, P. M., et al., Science 250:651 (1990); Hirata, R., et al., J. Biol. Chem. 265:6726 (1990); Cooper, A. and Stevens, T., TIBS 20:351 (1995); Xu, M. Q., et al., Cell 75:1371 (1993); Perler et al., Proc. Natl. Acad. Sci. USA 89:5577 (1992); Cooper et al., EMBO J. 225.75 (1993); Michel et al., Biochimie 64:867 (1992); Pietrokovski S., Prot. Sci. 3:2340 (1994). Most inteins in the M. jannaschii genome were identified by (i) similarity of the bounding exteins to other proteins, (ii) similarity of the inteins to those previously described, (iii) presence of the dodecapeptide endonuclease motifs, and (iv) canonical intein-exteinjunction sequences. In two instances (MJ0832 and MJ0043), the similarity to other database sequences did not unambiguously define the NH₂-terminal extein-intein junction, so it was necessary to rely on consensus sequences to select the putative site. The inteins in MJ1042 and MJ0542 have previously uricharacterized COOH-terminal splice junctions, GNC and FNC, respectively).

The sequences remaining after an intein is excised are called exteins, in analogy to exons. Exteins are spliced together after the excision of one or more inteins to form functional proteins. The biological significance and role of inteins are not clearly understood (Kane, P. M., et al., Science 250:651 (1990); Hirata, R., et al., J. Biol. Chem. 265:6726 (1990); Cooper, A. and Stevens, T., TIBS 20:351 (1995); Xu, M. Q., et al., Cell 75:1371 (1993); Perler et al., Proc. Natl. Acad. Sci. USA 89:5577 (1992); Cooper et al., EMBO J. 12:2575 (1993); Michel et al., Biochimie 64:867 (1992); Pietrokovski S., Prot. Sci. 3:2340 (1994)). Fourteen genes in the M. jannaschii genome contain 18 putative inteins, a significant increase in the approximately 10 intein-containing genes that have been described (Kane, P. M., et al., Science 250:651 (1990); Hirata, R., et al., J. Biol. Chem. 265:6726 (1990); Cooper, A. and Stevens, T., TIBS 20:351 (1995); Xu, M. Q., et al., Cell 75:1371 (1993); Perler et al., Proc. Natl. Acad. Sci. USA 89:5577 (1992); Cooper et al., EMBO J. 12:2575 (1993); Michel et al., Biochimie 64:867 (1992); Pietrokovski S., Prot. Sci. 3:2340 (1994)) (Table 4). The only previously described inteins in the Archaea are in the DNA polymerase genes of the Thermococcales (Kane, P. M., et al., Science 250:651 (1990); Hirata, R., et al., J. Biol. Chem. 265:6726 (1990); Cooper, A. and Stevens, T., TIBS 20:351 (1995); Xu, M. Q., et al., Cell 75:1371 (1993); Perler et al., Proc. Natl. Acad. Sci. USA 89:5577 (1992); Cooper et al., EMBO J. 12:2575 (1993); Michel et al., Biochimie 64:867 (1992); Pietrokovski S., Prot. Sci. 3:2340 (1994)). The M. jannaschi DNA polymerase gene has two inteins in the same locations as those in Pyrococcus sp. strain KOD1. In this case, the exteins exhibit 46% amino acid identity, whereas intein 2 of the two organisms has only 33% identity. This divergence suggests that intein 2 has not been recently (laterally) transferred between the Thermococcales and M. jannaschii. In contrast, the intein 1 sequences are 56% identical, more than that of the gene containing them, and comparable to the divergence of inteins within the Thermococcales. This high degree of sequence similarity might be the result of an intein transfer more recent than the splitting of these species. The large number of inteins found in M. jannaschii led us to question whether these inteins have been increasing in number by moving within the genome. If this were so, we would expect to find some pairs of inteins that are particularly similar. Comparisons of these and other available intein sequences showed that the closest relationships are those noted above linking the DNA polymerase inteins to correspondingly positioned elements in the Thermococcales. Within M. jannaschii, the highest identity observed was 33% for a 380-bp portion of two inteins. This finding suggests that the diversification of the inteins predates the divergence of the M. jannaschii and Pyrococcus DNA polymerases.

Three families of repeated genetic elements were identified in the M. jannaschii genome. Within two of the families, at least two members were identified as ORFs with a limited degree of sequence similarity to bacterial transposases. Members of the first family, designated ISAMJ1, are repeated 10 times on the main chromosome and once on the large ECE (FIG. 5). There is no sequence similarity between the IS elements in M. jannaschii and the ISM1 mobile element described previously for Methanobrevibacter smithii (Hamilton, P. T. et al., Mol. Gen. Genet. 200:47 (1985)). Two members of this family were identified as ORFs and are 27% identical (at the amino acid sequence level) to a transposase from Bacillus thuringiensis (IS240; GenBank accession number M23741). Relative to these two members, the remaining members of the ISAMJ1 family are missing an internal region of several hundred nucleotides (FIG. 5). With one exception, all members of this family end with 16-bp terminal inverted repeats typical of insertion sequences. One member is missing the terminal repeat at its 5′ end. The second family consists of two ORFs that are identical across 928 bp. The ORFs are 23% identical at the amino acid sequence level to the COOH-terminus of a transposase from Lactococcus lactis (IS982; GenBank accession number L34754). Neither of the members of the second family contains terminal inverted repeats.

Eighteen copies of the third family of repeated genetic structures (FIG. 6) are distributed fairly evenly around the M. jannaschii genome (FIG. 4). Unlike the genetic elements described above, none of the components of this repeat unit appears to have coding potential: The repeat structure is composed of a long segment followed by one to 25 tandem repetitions of a short segment. The short segments are separated by sequence that is unique within and among the complete repeat structure. Three similar types of short segments were identified; however, the type of short repeat is consistent within each repeat structure, except for variation of the last short segment in six repeat structures. Similar tandem repeats of short segments have been observed in Bacteria and other Archaea (Mojica, F. J. M., et al., Mol. Micro. 17:85 (1995)) and have been hypothesized to participate in chromosome partitioning during cell division.

The 16-kbp ECE from M. jannaschii contains 12 ORFs, none of which had a significant full-length match to any published sequence (FIG. 3). The 58-kbp ECE contains 44 predicted protein-coding regions, 5 of which had matches to genes in the database. Two of the genes are putative archaeal histones, one is a sporulation-related protein (SOJ protein), and two are type I restriction modification enzymes. There are several instances in which predicted protein-coding regions or repeated genetic elements on the large ECE have similar counterparts on the main chromosome of M. jannaschii (FIG. 3). The degree of nucleotide sequence similarity between genes present on both the ECE and the main chromosome ranges from 70 to 90%, suggesting that there has been relatively recent exchange of at least some genetic material between the large ECE and the main chromosome.

All the predicted protein-coding regions from M. jannaschii were searched against each other in order to identify families of paralogous genes (genes related by gene duplication, not speciation). The initial criterion for grouping paralogs was >30% amino acid sequence identity over 50 consecutive amino acid residues. Groups of predicted protein-coding regions were then aligned and inspected individually to ensure that the sequence similarity extended over most of their lengths. This curatorial process resulted in the identification of more than 100 gene families, half of which have no database matches. The largest identified gene family (16 members) (FIG. 7) contains almost 1% of the total predicted protein-coding regions in M. jannaschii.

Despite the availability for comparison of two complete bacterial genomes and several hundred megabase pairs of eukaryotic sequence data, the majority of genes in M. jannaschii cannot be identified on the basis of sequence similarity. Previous evidence for the shared common ancestry of the Archaeal and Eukaryotic was based on a small set gene sequences (Iwabe, N., et al., Proc. Natl. Acad. Sci. USA 86:9355 (1989); Gogarten J. P., et al., Proc. Natl. Acad. Sci. USA 86:6661 (1989); Brown, J. R. and Doolittle, W. F., Proc. Natl. Acad. Sci. USA 92:2441 (1995)). The complete genome of M. jannaschii allows us to move beyond a “gene by gene” approach to one that encompasses the larger picture of metabolic capacity and cellular systems. The anabolic genes of M. jannaschii (especially those related to energy production and nitrogen fixation) reveal an ancient metabolic world shared largely by Bacteria and Archaea. That many basic autotrophic pathways appear to have a common evolutionary origin suggests that the most recent universal common ancestor to all three domains of extant life had the capacity for autotrophy. The Archaea and Bacteria also share structural and organizational features that the most recent universal prokaryotic ancestors also likely possessed, such as circular genomes and genes organized as operons. In contrast, the cellular information-processing and secretion systems in M. jannaschii demonstrate the common ancestry of Eukaryotes and Archaea. Although there are components of these systems are present in all three domains, their apparent refinement over time-especially transcription and translation-indicate that the Archaea and Eukaryotes share a common evolutionary trajectory independent of the lineage of Bacteria.

EXAMPLE 2 Preparation of PCR Primers and Amplification of DNA

Various fragments of the Methanococcus jannaschii genome, such as those disclosed in Tables 2(a), 2(b) and 3 can be used, in accordance with the present invention, to prepare PCR primers. The PCR primers are preferably at least 15 bases, and more preferably at least 18 bases in length. When selecting a primer sequence, it is preferred that the primer pairs have approximately the same G/C ratio, so that melting temperatures are approximately the same. The PCR primers are useful during PCR cloning of the ORFs described herein.

EXAMPLE 3 Gene expression from DNA Sequences Corresponding to ORFs

A fragment of the Methanococcus jannaschii genome (preferably, a protein-encoding sequence) provided in Tables 2(a), 2(b) or 3 is introduced into an expression vector using conventional technology (techniques to transfer cloned sequences into expression vectors that direct protein translation in mammalian, yeast, insect or bacterial expression systems are well known in the art). Commercially available vectors and expression systems are available from a variety of suppliers including Stratagene (La Jolla, Calif.), Promega (Madison, Wis.), and Invitrogen (San Diego, Calif.). If desired, to enhance expression and facilitate proper protein folding, the codon context and codon pairing of the sequence may be optimized for the particular expression organism, as explained by Hatfield et al, U.S. Pat. No. 5,082,767, which is hereby incorporated by reference.

The following is provided as one exemplary method to generate polypeptide(s) from a cloned ORF of the Methanococcus genome whose sequence is provided in SEQ ID NOS:1, 2 and 3. A poly A sequence can be added to the construct by, for example, splicing out the poly A sequence from pSGS (Stratagene) using Bg/I and Sa/I restriction endonuclease enzymes and incorporating it into the mammalian expression vector pXT1 (Stratagene) for use in eukaryotic expression systems. pXT1 contains the LTRs and a portion of the gag gene from Moloney Murine Leukemia Virus. The position of the LTRs in the construct allow efficient stable transfection. The vector includes the Herpes Simplex thymidine kinase promoter and the selectable neomycin gene. The Methanococcus DNA is obtained by PCR from the bacterial vector using oligonucleotide primers complementary to the Methanococcus DNA and containing restriction endonuclease sequences for PstI incorporated into the 5′ primer and Bg/II at the 5′ end of the corresponding Methanococcus DNA 3′ primer, taking care to ensure that the Methanococcus DNA is positioned such that its followed with the poly A sequence. The purified fragment obtained from the resulting PCR reaction is digested with PstI, blunt ended with an exonuclease, digested with Bg/II, purified and ligated to pXT1, now containing a poly A sequence and digested Bg/II.

The ligated product is transfected into mouse NIH 3T3 cells using Lipofectin (Life Technologies, Inc., Grand Island, N.Y.) under conditions outlined in the product specification. Positive transfectants are selected after growing the transfected cells in 600 ug/ml G418 (Sigma, St. Louis, Mo.). The protein is preferably released into the supernatant. However if the protein has membrane binding domains, the protein may additionally be retained within the cell or expression may be restricted to the cell surface.

Since it may be necessary to purify and locate the transfected product, synthetic 15-mer peptides synthesized from the predicted Methanococcus DNA isequence are injected into mice to generate antibody to the polypeptide encoded by the Methanococcus DNA.

If antibody production is not possible, the Methanococcus DNA sequence is additionally incorporated into eukaryotic expression vectors and expressed as a chimeric with, for example, β-globin. Antibody to β-globin is used to purify the chimeric. Corresponding protease cleavage sites engineered between the β-globin gene and the Methanococcus DNA are then used to separate the two polypeptide fragments from one another after translation. One useful expression vector for generating β-globin chimerics is pSG5 (Stratagene). This vector encodes rabbit β-globin. Intron II of the rabbit β-globin gene facilitates splicing of the expressed transcript, and the polyadenylation signal incorporated into the construct increases the level of expression. These techniques as described are well known to those skilled in the art of molecular biology. Standard methods are available from the technical assistance representatives from Stratagene, Life Technologies, Inc., or Promega. Polypeptides may additionally be produced from either construct using in vitro translation systems such as In vitro Express™ Translation Kit (Stratagene).

EXAMPLE 4 E. coli Expression of a M. jannaschii ORF and protein purification

A M. jannaschii ORF described in Table 2(a), 2(b), or 3 is selected and amplified using PCR oligonucleotide primers designed from the nucleotide sequences flanking the selected ORF and/or from portions of the ORF's NH₂- or COOH-terminus. Additional nucleotides containing restriction sites to facilitate cloning are added to the 5′ and 3′ sequences, respectively.

The restriction sites are selected to be convenient to restriction sites in the bacterial expression vector pD10 (pQE9), which is used for bacterial expression. (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, Calif., 91311). [pD10]pQE9 encodes ampicillin antibiotic resistance (“Amp”) and contains a bacterial origin of replication (“ori”), an IPTG inducible promoter, a ribosome binding site (“RBS”), a 6-His tag and restriction enzyme sites.

The amplified M. jannaschii DNA and the vector pQE9 both are digested with SalI and XbaI and the digested DNAs are then ligated together. Insertion of the M. jannaschii DNA into the restricted pQE9 vector places the M. jannaschii coding region downstream of and operably linked to the vector's IPTG-inducible promoter and in-frame with an initiating AUG appropriately positioned for translation of the M. jannaschii protein.

The ligation mixture is transformed into competent E. coli cells using standard procedures. Such procedures are described in Sambrook et al., Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strain M15/rep4, containing multiple copies of the plasmid pREP4, which expresses lac repressor and confers kanamycin resistance (“Kan”), is used in carrying out the illustrative example described herein. This strain, which is only one of many that are suitable for expressing M. jannaschii protein, is available commercially from Qiagen.

Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by restriction analysis. Clones containing the desired constructs are grown overnight (“O/N”) in liquid culture in LB media supplemented with both ampicillin (100 μg/ml) and kanamycin (25 μg/ml).

The O/N culture is used to inoculate a large culture, at a dilution of approximately 1:100 to 1:250. The cells are grown to an optical density at 600 nm (“OD600”) of between 0.4 and 0.6. Isopropyl-B-D-thiogalactopyranoside (“IPTG”) is then added to a final concentration of 1 mM to induce transcription from lac repressor sensitive promoters, by inactivating the lacI repressor. Cells subsequently are incubated further for 3 to 4 hours. Cells then are harvested by centrifugation and disrupted, by standard methods. Inclusion bodies are purified from the disrupted cells using routine collection techniques, and protein is solubilized from the inclusion bodies into 8M urea. The 8M urea solution containing the solubilized protein is passed over a PD-10 column in 2×phosphate-buffered saline (“PBS”), thereby removing the urea, exchanging the buffer and refolding the protein. The protein is purified by a further step of chromatography to remove endotoxin followed by sterile filtration. The sterile filtered protein preparation is stored in 2×PBS at a concentration of 95 μ/ml.

EXAMPLE 5 Cloning and Expression of a M. jannaschii protein in a Baculovirus 25 Expression. System

A M. jannaschii ORF described in Table 2(a), 2(b), or 3 is selected and amplified as above. The amplified DNA is isolated from a 1% agarose gel using a commercially available kit (“Gene clean,” BIO 101 Inc., La Jolla, Calif.). The DNA then is digested with XbaI and again purified on a 1% agarose gel. This DNA is designated herein as F2.

The vector pA2-GP is used to express the M. jannaschii protein in the baculovirus expression system as described in Summers et al., A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987). The pA2-GP expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites. The signal peptide of AcMNPV gp67, including the N-terminal methionine, is located just upstream of a BamHI site. The polyadenylation site from the simian virus 40 (“SV40”) is used for efficient polyadenylation. For an easy selection of recombinant virus, the beta-galactosidase gene from E. coli is inserted in the same orientation as the polyhedrin promoter and is followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate viable virus that express the cloned polynucleotide.

Many other baculovirus vectors could be used in place of pA2-GP, such as pAc373, pVL941 and pAcIMI provided, as those of skill readily will appreciate, that construction provides appropriately located signals for transcription, translation, trafficking and the like, such as an in-frame AUG and a signal peptide, as required. Such vectors are described in Luckow et al., Virology 170: 31-39, among others.

The plasmid is digested with the restriction enzyme XbaI and then is dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.). This vector DNA is designated herein “V”.

Fragment F2 and the dephosphorylated plasmid V2 are ligated together with T4 DNA ligase. E. coli HB101 cells are transformed with ligation mix and spread on culture plates. Bacteria are identified that contain the plasmid with the M. jannaschii gene by digesting DNA from individual colonies using XbaI and then analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid is designated herein pBacM. jannaschii.

5 μg of the plasmid pBacM. jannaschii is co-transfected with 1.0 μg of a commercially available linearized baculovirus DNA (“BaculoGold™ baculovirus DNA”, Phanningen, San Diego, Calif.), using the lipofection method described by Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7417 (1987). 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBacM. jannaschii are mixed in a sterile well of a microtiter plate containing 50 μl of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is rocked back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27° C. After 5 hours the transfection solution is removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. The plate is put back into an incubator and cultivation is continued at 27° C. for four days.

After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, cited above. An agarose gel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a “plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10).

Four days after serial dilution, the virus is added to the cells. After appropriate incubation, blue stained plaques are picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruses is then resuspended in an Eppendorf tube containing 200 μl of Grace's medium. The agar is removed by a brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatant of these culture dishes are harvested and then they are stored at 4° C. A clone containing properly inserted hESSB I, II and III is identified by DNA analysis including restriction mapping and sequencing. This is designated herein as V-M. jannaschii.

Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus V-M. jannaschii at a multiplicity of infection (“MOI”) of about 2 (about 1 to about 3). Six hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Gaithersburg). 42 hours later, 5 μCi of ³⁵S-methionine and 5 μCi ³⁵S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then they are harvested by centrifugation, lysed and the labeled proteins are visualized by SDS-PAGE and autoradiography.

EXAMPLE 6 Cloning and Expression in Mammalian Cells

Most of the vectors used for the transient expression of a M. jannaschii gene in mammalian cells should carry the SV40 origin of replication. This allows the replication of the vector to high copy numbers in cells (e.g., COS cells) which express the T antigen required for the initiation of viral DNA synthesis. Any other mammalian cell line can also be utilized for this purpose.

A typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein-coding sequence, and signals required for the termination of trancription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular signals can also be used (e.g., human actin promoter). Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be used include, human Hela, 283, H9 and Jurkart cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, African green monkey cells, quail QC1-3 cells, mouse L cells and Chinese hamster ovary cells.

Alternatively, the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome. The co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) is a useful marker to develop cell lines that carry several hundred or even several thousand copies of the gene of interest. Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992)). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified genes(s) integrated into a chromosome. Chinese hamster ovary (CHO) cells are often used for the production of proteins.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al, Molecular and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, XbaI and Asp7l8, facilitate the cloning of the gene of interest. The vectors contain in addition the 3′ intron, the polyadenylation and termination signal of the rat preproinsulin gene.

EXAMPLE 6(a) Cloning and Expression in COS Cells

The expression plasmid, pM. jannaschii HA, is made by cloning a cDNA encoding a M. jannaschii protein into the expression vector pcDNAI/Amp (which can be obtained from Invitrogen, Inc.).

The expression vector pcDNAI/amp contains: (1) an E. coli origin of replication effective for propagation in E. coli and other prokaryotic cells; (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron, and a polyadenylation signal arranged so that a cDNA conveniently can be placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker.

A DNA fragment encoding the M. jannaschii protein and an HA tag fused in frame to its 3′ end is cloned into the polylinker region of the vector so that recombinant protein expression is directed by the CMV promoter. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson et al., Cell 37: 767 (1984). The fusion of the HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.

The PCR amplified DNA fragment (generated as described above) and the vector, pcDNAI/Amp, are digested with HindIII and XhoI and then ligated. The ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037), and the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis and gel sizing for the presence of the M. jannaschii protein-encoding fragment.

For expression of recombinant M. jannaschii, COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for instance, in Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, N.Y. (1989). Cells are incubated under conditions for expression of M. jannaschii protein by the vector.

Expression of the M. jannaschii HA fusion protein is detected by radiolabelling and immunoprecipitation, using methods described in, for example Harlow et al., Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To this end, two days after transfection, the cells are labeled by incubation in media containing ³⁵S-cysteine for 8 hours. The cells and the media are collected, and the cells are washed and the lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. cited above. Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody. The precipitated proteins then are analyzed by SDS-PAGE gels and autoradiography. An expression product of the expected size is seen in the cell lysate, which is not seen in negative controls.

EXAMPLE 6(b) Cloning and Expression in CHO Cells

The vector pC1 is used for the expression of a M. jannaschii protein. —Plasmid pC1 is a derivative of the plasmid pSV2-dhfr [ATCC Accession No. 37146]. Both plasmids contain the mouse DHFR gene under control of the SV40 early promoter. Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate. The amplification of the DHFR genes in cells resistant to methotrexate (MTX) has been well documented (see, e.g., Alt, F. W., Kellems, R. M., Bertino, J. R., and Schirnke, R. T., 1978, J. Biol. Chem. 253:1357-1370, Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta, 1097:107-143, Page, M. J. and Sydenham, M. A. 1991, Biotechnology Vol. 9:64-68). Cells grown in increasing concentrations of MTX develop resistance to the drug by overproducing the target enzyme, DHFR, as a result of amplification of the DHFR gene. If a second gene is linked to the DHFR gene it is usually co-amplified and over-expressed. It is state of the art to develop cell lines carrying more than 1,000 copies of the genes. Subsequently, when the methotrexate is withdrawn, cell lines contain the amplified gene integrated into the chromosome(s).

Plasmid pC1 contains for the expression of the gene of interest a strong promoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus (Cullen, et al., Molecular and Cellular Biology, March 1985:4384470) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al., Cell 41:521-530, 1985). Downstream of the promoter are the following single restriction enzyme cleavage sites that allow the integration of the genes: BamHI, Pvull, and Nrul. Behind these cloning sites the plasmid contains translational stop codons in all three reading flames followed by the 3′ intron and the polyadenylation site of the rat preproinsulin gene. Other high efficient promoters can also be used for the expression, e.g., the human β-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI. For the polyadenylation of the mRNA other signals, e.g., from the human growth hormone or globin genes can be used as well.

Stable cell lines carrying the gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate.

The plasmid pC1 is digested with the restriction enzyme BamHI and then dephosphorylated using calf intestinal phosphates by procedures known in the art. The vector is then isolated from a 1% agarose gel.

The M. jannaschii protein-encoding sequence is is amplified using PCR oligonucleotide primers as described above. An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J. Mol. Biol. 196:947-950 (1987) is appropriately located in the vector portion of the construct. The amplified fragments are isolated from a 1% agarose gel as described above and then digested with the endonucleases BamHI and Asp718 and then purified again on a 1% agarose gel.

The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 cells are then transformed and bacteria identified that contained the plasmid pC1 inserted in the correct orientation using the restriction enzyme BamHI. The sequence of the inserted gene is confirmed by DNA sequencing.

Transfection of CHO-DHFR-cells

Chinese hamster ovary cells lacking an active DHFR enzyme are used for transfection. 5 μg of the expression plasmid C1 are cotransfected with 0.5 μg of the plasmid pSVneo using the lipofecting method (Felgner et al., supra). The plasmid pSV2-neo contains a dominant selectable marker, the gene neo from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) and cultivated from 10-14 days. After this period, single clones are trypsinized and then seeded in 6-well petri dishes using different concentrations of methotrexate (25 nM, 50 nM, 100 nM, 200 nM, 400 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (500 nM, 1 μM, 2 μM, 5 μM). The same procedure is repeated until clones grow at a concentration of 100 μM.

The expression of the desired gene product is analyzed by Western blot analysis and SDS-PAGE.

EXAMPLE 7 Production of an Antibody to a Methanococcus jannaschii Protein

Substantially pure M. jannaschii protein or polypeptide is isolated from the transfected or transformed cells described above using an art-known method. The protein can also be chemically synthesized. Concentration of protein in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms/ml. Monoclonal or polyclonal antibody to the protein can then be prepared as follows:

Monoclonal Antibody Production by Hybridoma Fusion

Monoclonal antibody to epitopes of any of the peptides identified and isolated as described can be prepared from murine hybridomas according to the -classical method of Kohler, G. and Milstein, C., Nature 256:495 (1975) or modifications of the methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the'system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall, E., Meth. Enzymol. 70:419 (1980), and modified methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Davis, L. et al. Basic Methods in Molecular Biology Elsevier, New York. Section 21-2 (1989).

Polyclonal Antibody Production by Immunization

Polyclonal antiserum containing antibodies to heterogenous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein described above, which can be unmodified or modified to enhance immunogenicity. Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than other molecules and may require the use of carriers and adjuvant. Also, host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera Small doses (ng level) of antigen administered at multiple intradermal sites appears to be most reliable. An effective immunization protocol for rabbits can be found in Vaitukaitis, J. et al., J. Clin. Endocrinol. Metab. 33:988-991(1971).

Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall (See Ouchterlony, O. et al., Chap. 19 in: Handbook of Experimental Immunology, Wier, D., ed, Blackwell (1973)). Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12 _(μ)M). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher, D., Chap. 42 in: Manual of Clinical Immunology, second edition, Rose and Friedman, (eds.), Amer. Soc. For Microbio., Washington, D.C. (1980).

Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.

TABLE 1A Amino acid biosynthesis Aromatic amino acid family MJ1454 47830 48390 3-dehydroquinate dehydratase {Escherichia coli} 32.6 54.0 561 MJ0502 1029204 1027915 5-enolpyruvylshikimate 3-phosphate synthase {Haemophilus influenzae} 38.2 60.0 1290 MJ1075 456842 458158 anthranilate synthase, subunit I {Clostridium thermocellum} 52.7 72.1 1317 MJ0234 1247181 1246243 anthranilate synthase, subunit II′ {Thermotoga maritima} 44.1 64.3 939 MJ0238 1242410 1241916 anthranilate synthase, subunit II″ {Thermotoga maritima} 52.6 75.0 495 MJ0246 1238364 1238660 chorismate mutase subunit A {Erwinia herbicola} 37.4 59.4 297 MJ0612 929781 928723 chorismate mutase subunit B {Escherichia coli} 33.2 56.2 1059 MJ1175 357469 358572 chorismate synthase {Synechocystis sp} 48.8 66.5 1104 MJ0918 621924 622682 indole-3-glycerol phosphate synthase {Halobacterium volcanii} 42.7 67.7 759 MJ0451 1068501 1067845 N-phosphoribosyl anthranilate isomerase {Haloferax volcanii} 41.9 62.5 657 MJ0637 904569 905264 prephenate dehydratase {Lactococcus lactis} 39.3 61.7 696 MJ1084 449533 448757 shikimate 5-dehydrogenase {Escherichia coli} 38.9 57.4 777 MJ1038 502619 501777 tryptophan synthase, subunit alpha {Methanobacterium thermoautotrophicum} 49.8 69.3 843 MJ1037 503929 502808 tryptophan synthase, subunit beta {Acinetobacter calcoaceticus} 62.2 78.7 1122 Aspartate family MJ1116 414120 415679 asparagine synthetase {Escherichia coli} 34.0 54.3 1560 MJ1056 476613 476170 asparagine synthetase {Bacillus subtilis} 33.0 54.6 444 MJ1391 132691 133833 aspartate aminotransferase {Sulfolobus solfataricus} 31.0 52.2 1143 MJ0684 859565 860632 aspartate aminotransferase {Sulfolobus solfataricus} 37.8 63.7 1068 MJ0001 1469369 1470142 aspartate aminotransferase {Sulfolobus solfataricus} 39.2 63.8 774 MJ0205 1273947 1274951 aspartate-semialdehyde dehydrogenase {Leptospira interrogans} 50.4 67.2 1005 MJ0571 963902 962544 aspartokinase I {Serratia marcescens} 37.0 56.7 1359 MJ1473 26812 27558 cobalamin-independent methionine synthase {Methanobacterium thermoautotrophicum} 47.7 65.3 747 MJ1097 433957 435159 diaminopimelate decarboxylase {Haemophilus influenzae} 43.2 66.6 1203 MJ1119 412913 412029 diaminopimelate epimerase {Haemophilus influenzae} 36.2 56.6 885 MJ0422 1090629 1091441 dihydrodipicolinate reductase {Haemophilus influenzae} 45.0 64.4 813 MJ0244 1239093 1239776 dihydrodipicolinate synthase {Haemophilus influenzae} 46.6 64.4 684 MJ1003 540278 539106 homoaconitase {Saccharomyces cerevisiae} 35.7 56.9 1173 MJ1602 1563296 1562289 homoserine dehydrogenase {Bacillus subtilis} 40.4 63.2 1008 MJ1104 427241 428128 homoserine kinase {Haemophilus influenzae} 30.1 53.9 888 MJ0020 1450056 1451210 L-asparaginase I {Haemophilus influenzae} 34.8 53.1 1155 MJ0457 1064285 1063176 succinyl-diaminopimelate desuccinylase {Haemophilus influenzae} 27.0 45.8 1110 MJ1465 36982 38157 threoninesynthase {Bacillus subtilis} 51.2 71.1 1176 Glutamate family MJ0069 1406333 1405455 acetylglutamate kinase {Bacillus stearothermophilus} 44.4 65.7 879 MJ0791 757315 758637 argininosuccinate lyase {Campylobacter jejuni} 41.3 65.6 1323 MJ0429 1087105 1086023 argininosuccinate synthase {Methanococcus vannielii} 70.2 86.8 1083 MJ0186 1287178 1288140 glutamate N-acetyltransferase {Bacillus stearothermophilus} 47.4 63.1 963 MJ1351 172535 174007 glutamate synthase (NADPH), subunit alpha {Escherichia coli} 40.5 54.0 1473 MJ1346 179417 178068 glutamine synthetase {Methanococcus voltae} 70.5 84.7 1350 MJ1096 435486 436508 N-acetyl gamma-glutamyl-phosphate reductase {Bacillus subtilis} 40.4 63.6 1023 MJ0721 817148 816045 N-acetylornithine aminotransferase {Anabaena sp.} 46.7 67.0 1104 MJ0881 664952 665845 ornithine carbamoyltransferase {Halobacterium halobium} 43.0 69.6 894 Pyruvate family MJ0503 1027812 1026610 2-isopropylmalate synthase {Lactococcus lactis} 44.4 61.1 1203 MJ1392 131826 130633 2-isopropylmalate synthase {Anabaena sp.} 43.0 63.1 1194 MJ1271 256614 256216 3-isopropylmalate dehydratase {Salmonella typhimurium} 44.1 62.0 399 MJ1277 249421 249807 3-isopropylmalate dehydratase {Clostridium pasteurianum} 49.5 70.2 387 MJ0663 884580 883129 acetolactate synthase, large subunit {Porphyra umbilicalis} 34.5 54.6 1452 MJ0277 1207735 1209507 acetolactate synthase, large subunit {Bacillus subtilis} 50.2 69.7 1773 MJ0161 1307199 1307702 acetolactate synthase, small subunit {Bacillus subtilis} 49.4 74.1 504 MJ1008 533323 534132 branched-chain amino acid aminotransferase {Escherichia coli} 42.6 59.0 810 MJ1276 250052 251710 dihydroxy-acid dehydratase {Lactococcus lactis} 44.6 65.1 1659 MJ1195 333450 335003 isopropylmalate synthase {Haemophilus influenzae} 42.9 63.7 1554 MJ1543 1615932 1614931 ketol-acid reductoisomerase {Bacillus subtilis} 53.7 77.0 1002 Serine family MJ1597 1568671 1567445 glycine hydroxymethyltransferase {Methanobacterium thermoautotrophicum} 69.8 80.7 1227 MJ1018 523454 524806 phosphoglycerate dehydrogenase {Bacillus subtilis} 42.7 65.4 1353 MJ1594 1571545 1571039 phosphoserine phosphatase {Haemophilus influenzae} 40.4 62.7 507 MJ0959 580672 581778 serine aminotransferase {Methanobacterium thermoformicicum} 54.5 74.9 1107 Histidine family MJ1204 324063 324878 ATP phosphoribosyltransferase {Escherichia coli} 34.0 57.3 816 MJ1456 46532 45354 histidinol dehydrogenase {Lactococcus lactis} 47.6 67.5 1179 MJ0955 586179 585073 histidinol-phosphate aminotransferase {Bacillus subtilis} 37.7 60.8 1107 MJ0698 848921 848364 imidazoleglycerol-phosphate dehydrogenase {Methanobacterium thermoautotrophicum} 51.7 71.2 558 MJ0506 1024803 1025237 imidazoleglycerol-phosphate synthase (amidotransferase) {Lactococcus lactis} 45.6 62.1 435 MJ0411 1101451 1100636 imidazoleglycerol-phosphate synthase (cyclase) {Azospirillum brasilense} 61.5 78.8 816 MJ1430 71328 71047 phosphoribosyl AMP cyclohydrolase {Methanococcus vannielii} 70.0 86.3 282 MJ0302 1186990 1187208 phosphoribosyl-ATP pyrophosphohydrolase {Azotobacter chroococcum} 54.1 68.9 219 MJ1532 1628155 1627745 phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase 51.9 81.1 411 {Methanococcus thermolithotrophicus} Biosynthesis of cofactors, prosthetic groups, and carriers MJ0603 937289 938566 glutamate-1-semialdehyde aminotransferase {Bacillus subtilis} 51.7 70.6 1278 MJ0569 966316 967137 porphobilinogen deaminase {Bacillus subtilis} 41.2 61.4 822 MJ0493 1035991 1036839 quinolinate phosphoribosyltransferase {Escherichia coli} 39.3 61.6 849 MJ0407 1105699 1104965 quinolinate synthetase {Cyanophora paradoxa} 37.2 58.8 735 MJ1388 136484 135309 S-adenosylhomocysteine hydrolase {Sulfolobus solfataricus} 61.7 78.5 1176 Biotin MJ1297 227704 227021 6-carboxyhexanoate-CoA ligase {Bacillus sphaericus} 42.2 62.2 684 MJ1298 227005 225890 8-amino-7-oxononanoate synthase {Bacillus sphaericus} 44.4 64.8 1116 MJ1300 225025 223709 adenosylinethionine-8-amino-7-oxononanoate aminotransferase {Bacillus sphaericus} 39.9 64.2 1317 MJ1619 1543130 1543552 bifunctional protein {Haemophilus influenzae} 25.7 54.9 423 MJ1296 228286 228843 biotin synthetase {Bacillus sphaericus} 38.2 62.5 558 MJ1299 225741 225100 dethiobiotin synthetase {Bacillus sphaericus} 37.0 59.0 642 Heme and porphyrin MJ1438 66330 65833 cobalamin (5′-phosphate) synthase {Escherichia coli} 26.1 48.7 498 MJ0552 983686 984417 cobalamin biosynthesis J protein {Salmonella typhimurium} 26.7 51.2 732 MJ1314 212528 211842 cobalamin biosynthesis protein D {Pseudomonas denitrificans} 38.0 61.0 687 MJ0022 1448163 1447273 cobalamin biosynthesis protein D {Salmonella typhimurium} 35.5 61.1 891 MJ1569 1592308 1591700 cobalamin biosynthesis protein M {Salmonella typhimurium} 29.5 54.7 609 MJ1091 442661 443239 cobalamin biosynthesis protein M {Salmonella typhimurium} 53.7 74.4 579 MJ0908 635150 631647 cobalamin biosynthesis protein N {Pseudomonas denitrificans} 37.5 57.6 3504 MJ0484 1046784 1045324 cobyric acid synthase {Methanococcus voltae} 73.7 89.8 1461 MJ1421 85381 86352 cobyrinic acid a,c-diamide synthase {Salmonella typhimurium} 32.1 55.0 972 MJ0143 1332080 1330965 glutamyl-tRNA reductase {Methanobacterium thermoautotrophicum} 47.8 66.9 1116 MJ0643 899800 898910 porphobilinogen synthase {Methanothermus sociabilis} 62.5 79.9 891 MJ0930 612059 611430 precorrin isomerase {Salmonella typhimurium} 38.7 62.0 630 MJ0771 780420 779932 precorrin-2 methyltransferase {Salmonella typhimurium} 30.4 55.9 489 MJ0813 734876 735547 precorrin-3 methylase {Salmonella typhimurium} 44.2 68.4 672 MJ1578 1583277 1582501 precorrin-3 methylase {Salmonella typhimurium} 54.6 76.5 777 MJ1522 1637017 1636385 precorrin-6Y methylase {Salmonella typhimurium} 30.6 52.3 633 MJ0391 1116729 1117202 precorrin-8W decarboxylase {Salmonella typhimurium} 23.9 49.1 474 MJ0965 573234 572509 uroporphyrin-III C-methyltransferase {Bacillus megaterium} 54.7 72.5 726 MJ0994 549022 549444 uroporphyrinogen III synthase {Bacillus subtilis} 27.8 49.4 423 Menaquinone and ubiquinone MJ1645 1509624 1508923 coenzyme PQQ synthesis protein III {Haemophilus influenzae} 32.2 53.3 702 Molybdopterin MJ0824 725986 726762 molybdenum cofactor biosynthesis moaA protein {Haemophilus influenzae} 30.0 57.3 777 MJ0167 1301836 1302162 molybdenum cofactor biosynthesis moaB protein {Escherichia coli} 46.4 69.6 327 MJ1135 396359 396781 molybdenum cofactor biosynthesis moaC protein {Haemophilus influenzae} 49.2 70.9 423 MJ0886 654158 656017 molybdenum cofactor biosynthesis moeA protein {Escherichia coli} 34.5 55.2 1860 MJ0666 879771 880943 molybdenum cofactor biosynthesis moeA protein {Haemophiius influenzae} 33.6 56.4 1173 MJ1663 1491265 1490831 molybdopterin-guanine dinucleotide biosynthesis protein A {Escherichia coli} 27.7 48.0 435 MJ1324 197777 197076 molybdopterin-guanine dinucleotide biosynthesis protein B {Escherichia coli} 32.2 57.7 702 Pantothenate MJ0913 626982 627779 pantothenate metabolism flavoprotein {Haemophilus influenzae} 34.1 55.7 798 Riboflavin MJ0055 1416688 1417278 GTP cyclohydrolase II {Bacillus subtilis} 35.8 56.0 591 MJ0671 874773 875396 riboflavin-specific deaminase {Actinobacillus pleuropneumoniae} 43.0 65.3 624 Thioredoxin, glutaredoxin, and glutathione MJ1536 1622694 1623533 thioredoxin reductase {Mycoplasma genitalium} 38.5 58.0 840 MJ0530 1005917 1005420 thioredoxin-2 {Saccharomyces cerevisiae} 33.0 63.3 498 MJ0307 1184114 1184332 thioredoxin/glutaredoxin {Methanobacterium thermoautotrophicum} 48.7 69.5 219 Thiamine MJ1026 514172 515440 thiamine biosynthesis protein {Bacillus subtilis} 45.0 66.1 1269 MJ0601 940113 939400 thiamine biosynthetic enzyme {Zea mays} 35.1 53.0 714 Pyridine nucleotides MJ1352 170567 171163 NH(3)-dependent NAD+ synthetase {Mycoplasma genitalium} 47.5 63.8 597 Cell envelope Membranes, lipoproteins, and porins MJ0544 989805 990443 dolichyl-phosphate mannose synthase {Trypanosoma brucei} 35.1 57.1 639 MJ1057 475508 474981 glycosyl transferase {Neisseria gonorrhoeae} 25.8 50.0 528 MJ0611 931098 930679 membrane protein {Saccharum sp.} 50.0 57.2 420 MJ0827 724322 723900 membrane protein {Homo sapiens} 44.9 67.0 423 Murein sacculus and peptidoglycan MJ1160 371691 370390 amidase {Moraxella catarrhalis} 24.6 36.1 1302 MJ0204 1276277 1275219 amidophosphoribosyltransferase {Bacillus subtillis} 52.0 72.9 1059 Surface polysaccharides, lipopolysaccharides and antigens MJ0924 617598 618035 capsular polysaccharide biosynthesis protein {Staphylococcus aureus} 31.3 46.9 438 MJ1061 469649 470293 capsular polysaccharide biosynthesis protein D {Staphylococcus aureus} 56.3 72.2 645 MJ1055 478643 477735 capsular polysaccharide biosynthesis protein I {Staphylococcus aureus} 50.7 74.4 909 MJ1059 472326 471904 capsular polysaccharide biosynthsis protein M {Staphylococcus aureus} 34.4 55.0 423 MJ1607 1555624 1554455 LPS biosynthesis related rfbu-protein {Haemophilus influenzae} 33.4 57.6 1170 MJ1113 417528 418352 N-acetylglucosamine-1-phosphate transferase {Sulfolobus acidocaldarius} 29.9 57.9 825 MJ0399 1110873 1112204 phosphomannomutase {Vibrio cholerae} 37.0 57.8 1332 MJ1068 462901 464265 putative O-antigen transporter {Shigella flexneri} 24.5 46.6 1365 MJ1066 464369 465430 spore coat polysaccharide biosynthesis protein C {Bacillus subtillis} 55.3 75.8 1062 MJ1065 465444 466454 spore coat polysaccharide biosynthesis protein E {Bacillus subtillis} 37.9 59.0 1011 MJ1063 467331 467828 spore coat polysaccharide biosynthesis protein F {Bacillus subtillis} 36.0 55.4 498 MJ1062 467870 469279 spore coat polysaccharide biosynthesis protein G {Bacillus subtillis} 32.0 54.5 1410 MJ0211 1269601 1268732 UDP-glucose 4-epimerase {Streptococcus thermophilus} 35.1 54.8 870 MJ1054 481027 478712 UDP-glucose dehydrogenase {Xanthomonas campestris} 42.8 63.4 2316 MJ0428 1087456 1088655 UDP-N-acetyl-D-mannosaminuronic acid dehydrogenase {Escherichia coli} 45.1 68.2 1200 Surface structures MJ0891 650616 650005 flagellin 31 {Methanococcus voltae} 55.4 71.6 612 MJ0892 649880 649269 flagellin 32 {Methanococcus voltae} 61.1 78.4 612 MJ0893 649163 648516 flagellin 33 {Methanococcus voltae} 59.1 78.7 648 Cellular processes Cell division MJ1489 10595 8721 cell division control protein {Saccharomyces cerevisiae} 34.8 57.7 1875 MJ0363 1142460 1140220 cell division control protein 21 {Schizosaccharomyces pombe} 30.0 51.4 2241 MJ1156 375317 377947 cell division control protein CDC48 {Saccharomyces cerevisiae} 51.9 71.7 2631 MJ0169 1300988 1300329 cell division inhibitor {Bacillus subtillis} 28.8 51.2 660 MJ0579 957291 958088 cell division inhibitor {Bacillus subtillis} 31.8 53.2 798 MJ0547 988025 988732 cell division inhibitor {Bacillus subtillis} 32.8 57.7 708 MJ0084 1393471 1392869 cell division inhibitor minD {Escherichia coli} 32.1 50.4 603 MJ0174 1295971 1294976 cell division protein {Drosophila melanogaster} 28.4 54.6 996 MJ0370 1135876 1134956 cell division protein ftsZ {Anabaena 7120} 50.7 71.7 921 MJ1376 147975 147343 cell division protein J {Haemophilus influenzae} 39.8 58.5 633 MJ0622 920029 921168 cell division protein Z {Haloferax volcanii} 51.0 71.7 1140 MJ0148 1326798 1327538 centromere/microtubule-binding protein {Saccharomyces cerevisiae} 42.7 64.7 741 MJ1647 1508164 1507907 DNA binding protein {Methanococcus voltae} 54.7 80.3 258 MJ1643 1513857 1510351 P115 protein {Mycoplasma hyorhinis} 30.3 55.4 3507 Chaperones MJ0999 543921 545471 chaperonin {Methanopyrus kandleri} 73.5 87.6 1551 MJ0285 1202058 1202459 heat shock protein {Clostridium acetobutylicum} 29.0 44.6 402 MJ0278 1207276 1207548 rotamase, peptidyl-prolyl cis-trans isomerase {Haemophilus influenzae} 40.7 60.5 273 MJ0825 725091 725765 rotamase, peptidyl-prolyl cis-trans isomerase {Pseudomonas fluorescens} 31.8 60.8 675 Detoxification MJ0736 804803 805453 alkyl hydroperoxide reductase {Sulfolobus solfataricus} 66.1 84.8 651 MJ1541 1618786 1619868 N-ethylammeline chlorohydrolase {Rhodococcus rubropertinctus} 29.2 56.3 1083 Protein and peptide secretion MJ0478 1051985 1056078 preprotein translocase secY {Methanococcus vannielii} 70.9 88.8 1308 MJ0111 1365253 1364216 protein-export membrane protein {Streptomyces coelicolor} 25.9 51.7 1038 MJ1253 276673 277377 protein-export membrane protein {Escherichia coli} 30.5 57.0 705 MJ0260 1226090 1226644 signal peptidase {Canis familiaris} 32.6 54.5 555 MJ0101 1376106 1377308 signal recognition particle protein {Haemophilus influenzae} 42.0 61.6 1203 MJ0291 1198470 1197244 signal recognition particle protein {Sulfolobus acidocaldarius} 48.3 69.4 1227 Transformation MJ0781 768702 770798 klbA protein {Plasmid RK2} 34.6 54.9 2097 MJ0940 602402 601929 transformation sensitive protein {Homo sapiens} 35.0 53.9 474 Cellular processes MJECL17 20110 19889 archaeal histone {Pyrococcus sp.} 58.8 81.0 221 MJECL29 36456 26220 archaeal histone {Pyrococcus sp.} 64.2 83.6 236 MJ1258 271686 271486 archaeal histone {Pyrococcus sp.} 71.7 83.6 201 MJ0168 1301348 1301548 archaeal histone {Pyrococcus sp.} 67.2 86.6 201 MJ0932 610153 609953 archaeal histone {Pyrococcus sp.} 67.2 86.6 201 Central intermediary metabolism Amino sugars MJ1420 90244 86939 glutamine-fructose-6-phosphate transaminase {Escherichia coli} 41.2 61.5 3306 Degradation of polysaccharides MJ1611 1550816 1549542 alpha-amylase {Pyrococcus furiosus} 27.0 50.5 1275 MJ0555 981500 980529 endoglucanase {Homo sapiens} 44.1 66.8 972 MJ1610 1551992 1550967 glucoamylase {Clostridium sp} 28.0 49.2 1026 Other MJ1656 1498675 1497965 2-hydroxyhepta-2,4-diene-1,7-dioate isomerase {Escherichia coli} 40.2 61.6 711 MJ0406 1106800 1105907 ribokinase {Escherichia coli} 23.2 46.3 894 MJ0309 1182259 1183077 ureohydrolase {Methanothermus fervidus} 40.9 60.7 819 Phosphorus compounds MJ0963 575418 577049 N-methylhydantoinase {Arthrobacter sp.} 32.6 53.0 1632 MJ0964 573516 575345 N-methylhydantoinase {Arthrobacter sp.} 37.7 56.4 1830 Polyamine biosynthesis MJ0535 1001006 1002031 acetylpolyamine aminohydolase {D01044 Mycoplana} 33.3 48.6 1026 MJ0313 1179250 1179801 spermidine synthase {Homo sapiens} 32.3 57.7 552 Polysaccharides-(cytoplasmic) MJ1606 1555858 1551354 glycogen synthase {Hordeum vulgare} 33.7 58.3 1497 Nitrogen metabolism MJ1187 345237 344335 ADP-ribosylglycohydrolase (draG) {Rhodospirillum rubrum} 29.8 50.8 903 MJ0713 824113 826278 hydrogenase accessory protein {Azotobacter chroococcum} 33.8 54.8 2166 MJ0214 1267658 1267314 hydrogenase accessory protein {Azotobacter chroococcum} 30.7 56.5 345 MJ0676 869311 870276 hydrogenase expression/formation protein {Rhizobium leguminosarum} 46.1 65.3 966 MJ0442 1075480 1076028 hydrogenase expression/formation protein B {Rhizobium leguminosarum} 44.6 64.0 549 MJ0200 1279494 1279739 hydrogenase expression/formation protein C {Azotobacter vinelandii} 40.0 68.8 246 MJ0993 549539 550525 hydrogenase expression/formation protein D {Alcaligenes eutrophus} 44.7 63.5 987 MJ0631 914544 914089 hydrogenase maturation protease {Escherichia coli} 33.9 58.9 456 MJ1093 441468 440584 nifB protein {Anabaena sp} 43.1 67.2 885 MJ0879 667622 666984 nitrogenase reductase {Methanococcus voltae} 77.2 89.1 639 MJ0685 859442 858696 nitrogenase reductase related protein {Clostridium pasteurianum} 31.7 49.6 747 MJ1051 483344 484411 nodulation factor production protein {Bradyrhizobium japonicum} 32.1 51.1 1068 MJ1058 473947 473141 nodulation factor production protein {Bradyrhizobium japonicum} 37.7 58.0 807 Carbon Fixation MJ0152 1325036 1322820 carbon monoxide dehydrogenase, alpha subunit {Clostridium thermoaceticum} 42.1 65.6 2217 MJ0153 1322553 1320256 carbon monoxide dehydrogenase, alpha subunit {Methanothrix soehngenii} 47.9 67.3 2298 MJ0156 1319256 1317883 carbon monoxide dehydrogenase, alpha subunit {Clostridium thermoaceticum} 47.8 69.5 1374 MJ0728 809951 811783 carbon monoxide dehydrogenase, beta subunit {Rhodospirillum rubrum} 35.9 55.0 1833 MJ0112 1362285 1363667 corrinoid/iron sulfur protein, large subunit {Clostridium thermoaceticum} 32.9 55.1 1383 MJ0113 1361128 1362030 corrinoid/iron sulfur protein, small subunit {Clostridium thermoaceticum} 37.7 58.8 903 MJ1235 292453 293673 ribulose bisphosphate carboxylase, large subunit {Synechococcus sp} 42.4 60.3 1221 Energy metabolism Aerobic MJ0649 896262 894919 NADH oxidase {Enterococcus faecalis} 28.0 50.4 1344 MJ0520 1011104 1011892 NADH-ubiquinone oxidoreductase, subunit 1 {Paracentrotus lividus} 29.5 53.9 789 Anaerobic MJ0092 1385748 1384282 fumarate reductase {Thermoplasma acidophilum} 40.2 57.0 1467 ATP-proton motive force interconversion MJ0217 1263468 1265171 ATP synthase, subunit A {Enterococcus hirae} 60.3 76.6 1704 MJ0216 1265356 1266615 ATP synthase, subunit B {Methanosarcina barkeri} 69.4 84.5 1260 MJ0219 1261985 1263040 ATP synthase, subunit C {Haloferax volcanii} 28.1 50.0 1056 MJ0615 926124 926663 ATP synthase, subunit D {Enterococcus hirae} 34.8 56.8 540 MJ0220 1261297 1261737 ATP synthase, subunit E {Methanosarcina mazeii} 29.0 50.0 441 MJ0218 1263054 1263347 ATP synthase, subunit F {Haloferax volcanii} 21.5 52.1 294 MJ0222 1258252 1260294 ATP synthase, subunit I {Enterococcus hirae} 27.6 52.2 2043 MJ0221 1260641 1261060 ATP synthase, subunit K {Enterococcus hirae} 34.6 59.8 420 Electron transport MJ1446 57416 56646 cytochrome-c3 hydrogenase, gamma chain {Pyrococcus furiosus} 40.1 52.4 771 MJ0741 803000 803320 desulfoferrodoxin {Desulfovibrio vulgaris} 44.0 59.4 321 MJ0578 958094 958900 ferredoxin {Clostridium sticklandii} 49.1 56.9 807 MJ0061 1411998 1411759 ferredoxin {Methanococcus thermolithotrophicus} 42.9 59.0 240 MJ0722 815808 816038 ferredoxin {Methanobacterium thermoautotrophicum} 42.3 60.6 231 MJ0099 1379076 1379456 ferredoxin {Desulfovibrio desulfuricans} 40.0 62.0 381 MJ0199 1279976 1279791 ferredoxin {Methanococcus thermolithotrophicus} 74.6 84.8 186 MJ0533 1003408 1003575 ferredoxin 2[4Fe—4S] homolog {Methanosarcina thermophila} 36.9 54.4 168 MJ0624 918981 918808 ferredoxin 2[4Fe═4S] {Methanosarcina thermophila} 48.0 68.0 174 MJ0267 1217567 1218463 ferredoxin oxidoreductase, alpha subunit {Klebsiella pneumoniae} 29.4 50.2 897 MJ0276 1209645 1210727 ferredoxin oxidoreductase, alpha subunit {Halobacterium halobium} 44.5 63.0 1083 MJ0266 1218644 1219387 ferredoxin oxidoreductase, beta subunit {Klebsiella pneumoniae} 32.6 51.0 744 MJ0537 998693 999424 ferredoxin oxidoreductase, beta subunit {Halobacteriuin halobiuin} 41.3 61.1 732 MJ0268 1217015 1217272 ferredoxin oxidoreductase, delta subunit {Pyrococcus furiosus} 58.9 71.8 258 MJ0536 999441 999980 ferredoxin oxidoreductase, gamma subunit {Pyrococcus furiosus} 32.0 50.9 540 MJ0269 1216601 1216993 ferredoxin oxidoreductase, gamma subunit {Pyrococcus furiosus} 55.6 74.7 393 MJ0732 806970 808100 flavoprotein {Methanobacterium thermoautotrophicum} 40.4 62.3 1131 MJ1192 339066 338095 methylviologen-reducing hydrogenase, alpha chain {Methanococcus voltae} 75.0 88.6 972 MJ1191 340221 339385 methylviologen-reducing hydrogenase, gamma chain {Methanococcus voltae} 71.5 83.3 837 MJ1362 160414 161055 NADH deydrogenase, subunit 1 {Mitochondrion Oncorhynchus} 23.1 50.0 642 MJ0514 1016474 1017223 polyferredoxin {Methanococcus voltae} 36.7 52.5 750 MJ0934 608147 607521 polyferredoxin {Methanothermus fervidus} 40.9 54.3 627 MJ1303 220214 221701 polyferredoxin {Methanobacterium thermoautotrophicum} 39.5 56.1 1488 MJ1193 337655 336591 polyferredoxin {Methanococcus voltae} 61.7 74.5 1065 MJ1227 301853 301257 pyruvate formate-lyase activating enzyme {Clostridium pasteurianum} 31.4 50.0 597 MJ0735 805546 805785 rubredoxin {Clostridium thermosaccharolyticum} 59.7 77.0 240 MJ0740 803522 803659 rubredoxin {Clostridium thermosaccharolyticum} 64.5 84.5 138 Fermentation MJ0007 1463447 1462359 2-hydroxyglutaryl-CoA dehydratase, subunit beta {Acidaminococcus fermentans} 22.6 48.2 1089 Gluconeogenesis MJ1479 22527 21358 alanine aminotransferase 2 {Panicum miliaceum} 30.1 50.0 1170 MJ0542 991264 994794 phosphoenolpyruvate synthase {Pyrococcus furiosus} 60.3 78.3 3531 Glycolysis MJ1482 18946 18044 2-phosphoglycerate kinase {Methanothermus fervidus} 47.1 70.9 903 MJ0641 901393 902325 3-phosphoglycerate kinase {Methanothermus fervidus} 58.2 78.1 933 MJ0232 1248239 1249432 enolase {Bacillus subtilis} 57.7 78.2 1194 MJ1605 1557395 1558597 glucose-6-phosphate isomerase {Bacillus stearothermophilus} 32.3 54.6 1203 MJ1146 386093 387055 glyceraldehyde 3-phosphate dehydrogenase {Methanothermus fervidus} 59.5 77.6 963 MJ0490 1038560 1037697 lactate dehydrogenase {Thermotoga maritima} 39.9 63.2 864 MJ1411 100555 99167 NADP-dependent glyceraldehyde-3-phosphate dehydrogenase {L15191 Streptococcus} 39.2 59.6 1389 MJ0108 1367951 1366716 pyruvate, kinase {Bacillus stearothermophilus} 39.1 60.5 1236 MJ1528 1631071 1631589 triosephosphate isomerase {Mycoplasma genitalium} 29.0 49.1 519 Pentose phosphate pathway MJ0680 865484 866083 pentose-5-phosphate-3-epimerase {Solanum tuberosum} 44.2 62.5 600 MJ1603 1560724 1560047 ribose 5-phosphate isomerase {Mus musculus} 42.0 63.4 678 MJ0960 580121 580576 transaldolase {Bacillus subtilis} 60.7 79.5 456 MJ0681 864603 865355 transketolase′ {Homo sapiens} 43.7 58.5 753 MJ0679 866375 867073 transketolase″ {Homo sapiens} 36.0 61.3 699 Pyruvate dehydrogenase MJ0636 906464 905292 dihydrolipoamide dehydrogenase {Haloferax volcanii} 28.9 51.0 1173 Sugars MJ1418 91211 90669 fuculose-1-phosphate aldolase {Haemophilus influenzae} 29.1 48.7 543 TCA cycle MJ0499 1031331 1032530 aconitase {Saccharomyces cerevisiae} 29.7 49.8 1200 MJ1294 229770 230381 fumarate hydratase, class I′ {Bacillus stearothermophilus} 35.1 55.7 612 MJ0617 925239 924778 fumarate hydratase, class I″ {Bacillus stearothermophilus} 43.8 66.0 462 MJ1596 1568967 1569998 isocitrate dehydrogenase {Thermus aquaticus} 42.9 61.4 1032 MJ0720 817433 818431 isocitrate dehydrogenase (NADP) {Thermus aquaticus} 48.0 64.7 999 MJ1425 77051 76299 malate dehydrogenase {Methanothermus fervidus} 61.3 77.6 753 MJ0033 1438609 1437116 succinate dehydrogenase, flavoprotein subunit {Escherichia coli} 41.8 58.1 1494 MJ1246 282664 283449 succinyl-CoA synthetase, alpha subunit {Escherichia coli} 59.6 74.8 786 MJ0210 1271318 1270227 succinyl-CoA synthetase, beta subunit {Thermus aquaticus} 48.8 68.7 1092 Methanogenesis MJ0253 1232773 1232405 8-hydroxy-5-deazaflavin-reducing hydrogenase, delta subunit 47.1 71.0 369 {Methanobacterium thermoautotrophicum} MJ1035 505234 506022 coenzyme F420-dependent N5,N10-methylene-tetrahydromethanopterin dehydrogenase 66.5 79.8 789 {Methanobacterium thermoautotrophicum} MJ0727 811895 812725 coenzyme F420-reducing hydrogenase, alpha subunit {Methanobacterium 26.8 45.8 831 thermoautotrophicum} MJ0029 1442517 1441279 coenzyme F420-reducing hydrogenase, alpha subunit {Methanococcus voltae} 50.3 66.1 1239 MJ0030 1441022 1440558 coenzyme F420-reducing hydrogenase, alpha subunit {Methanococcus voltae} 66.5 83.3 465 MJ1349 175566 176222 coenzyme F420-reducing hydrogenase, beta subunit {Methanococcus voltae} 36.6 55.7 657 MJ0725 813779 814453 coenzyme F420-reducing hydrogenase, beta subunit {Methanobacterium 41.0 62.0 675 thermoautotrophicum} MJ0870 677657 679372 coenzyme, F420-reducing hydrogenase, beta subunit {Methanobacterium 42.7 63.2 1716 thermoautotrophicum} MJ0032 1439835 1438990 coenzyme F420-reducing hydrogenase, beta subunit {Methanococcus voltae} 72.0 85.5 846 MJ0726 812987 813499 coenzyme F420-reducing hydrogenase, gamma subunit {Methanococcus voltae} 42.7 59.4 513 MJ0031 1440505 1439873 coenzyme F420-reducing hydrogenase, gamma subunit {Methanococcus voltae} 75.5 87.3 633 MJ0295 1192687 1193304 formate dehydrogenase (fdhD) {Wolinella succinogenes} 35.6 57.7 618 MJ0006 1463887 1465020 formate dehydrogenase, alpha subunit {Methanobacterium formicicum} 41.6 61.1 1134 MJ1353 168767 170344 formate dehydrogenase, alpha subunit {Methanobacterium formicicum} 54.2 70.9 1578 MJ0005 1465405 1466247 formate dehydrogenase, beta subunit {Methanobacterium formicicum} 49.5 72.1 843 MJ0155 1319767 1319315 formate dehydrogenase, iron-sulfur subunit {Wolinella succinogenes} 41.7 56.9 453 MJ0264 1220122 1220433 formate hydrogenlyase, subunit 2 {Escherichia coli} 42.9 59.8 312 MJ0265 1219502 1219930 formate hydrogenlyase, subunit 2 {Escherichia coli} 45.5 61.0 429 MJ0515 1013710 1014735 formate hydrogenlyase, subunit 5 {Escherichia coli} 31.0 51.1 1026 MJ1027 514001 512871 formate hydrogenlyase, subunit 5 {Escherichia coli} 34.3 53.3 1131 MJ1363 159614 160018 formate hydrogenlyase, subunit 7 {Escherichia coli} 38.4 60.9 405 MJ0516 1013157 1013600 formate hydrogenlyase, subunit 7 {Escherichia coli} 48.8 65.6 444 MJ0318 1175065 1175823 formylmethanofuran:tetrahydromethanopterin formyltransferase 68.6 84.5 759 {Methanobacterium thermoautotrophicum} MJ1338 185930 185007 H(2)-dependent methylenetetrahydromethanopterin dehydrogenase related protein 29.1 50.5 924 {Methanobacterium thermoautotrophicum} MJ0715 823334 822423 H2-forming N5,N10-methylene-tetrahydromethanopterin dehydrogenase-related protein 29.9 52.5 912 {Methanococcus voltae} MJ0784 765279 764272 H2-forming N5,N10-methylene-tetrahydromethanopterin dehydrogenease 73.6 85.5 1008 {Methanococcus voltae} MJ1190 342199 341003 heterodisulfide reductase, subunit A {Methanobacterium thermoautotrophicum} 58.0 75.2 1197 MJ0743 801736 802422 heterodisulfide reductase, subunit B {Methandbacterium thermoautotrophicum} 59.3 79.0 687 MJ0863 684944 685798 heterodisulfide reductase, subunit B {Methanobacterium thermoautotrophicum} 63.2 80.2 855 MJ0744 801103 801489 heterodisulfide reductase, subunit C {Methanobacterium thermoautotrophicum} 53.4 68.4 387 MJ0864 684283 684840 heterodisulfide reductase, subunit C {Methanobacterium thermoautotrophicum} 52.6 69.9 558 MJ0118 1357167 1356667 methyl coenzyme M reductase II operon, protein D {Methanothermus fervidus} 53.2 77.5 501 MJ0083 1395319 1393880 methyl coenzyme M reductase II, alpha subunit {Methanothermus fervidus} 89.8 95.5 1440 MJ0081 1397700 1396351 methyl coenzyme M reductase II, beta subunit {Methanothermus fervidus} 79.7 89.4 1350 MJ0082 1396335 1395538 methyl coenzyme M reductase II, gamma subunit {Methanothermus fervidus} 83.0 92.1 798 MJ0844 702037 701465 methyl coenzyme M reductase operon, protein C {Methanococcus vannielii} 82.5 92.6 573 MJ0843 702395 702069 methyl coenzyme M reductase operon, protein D {Methanococcus voltae} 58.0 81.4 327 MJ1662 1491537 1493201 methyl coenzyme M reductase system, component A2 {Methanobacterium 37.1 60.1 1665 thermoauotrophicum} MJ1242 284878 286338 methyl coenzyme M reductase system, component A2 {Methanobacterium 60.9 77.8 1461 thermoautotrophicum} MJ0846 700322 698880 methyl coenzyme M reductase, alpha subunit {Methanococcus voltae} 86.1 92.1 1443 MJ0842 703907 702576 methyl coenzyme M reductase, beta subunit {Methanococcus vannielii} 75.3 87.4 1332 MJ0845 701389 700673 methyl coenzyme M reductase, gamma subunit {Methanococcus vannielii} 78.7 91.3 717 MJ1636 1520054 1519128 N5,N10-methenyl-tetrahydromethanopterin cyclohydrolase {Methanobacterium 69.6 82.3 927 thermdautotrophicum} MJ1534 1625526 1624534 N5,N10-methylene tetrahydromethanopterin reductase {Methanobacterium 66.2 79.7 993 thermoautotrophicum} MJ0850 696203 695895 N5-methyl-tetrahydromethanopterin:coenzyme M methyltransferase 36.6 59.8 309 {Methanobacterium thermoautotrophicum} MJ0849 696884 696216 N5-methyl-tetrahydromethanopterin:coenzyme M methyltransferase 41.8 62.3 669 {Methanobacterium thermoautotrophicum} MJ0852 695117 694914 N5-methyl-tetrahydromethanopterin:coenzyme M methyltransferase 37.1 64.6 204 {Methanobacterium thermoautotrophicum} MJ0851 695866 695138 N5-methyl-tetrahydromethanopterin:coenzyme M methyltransferase 55.2 73.5 729 {Methanobacterium thermoautotrophicum} MJ0847 698519 697749 N5-methyl-tetrahydromethanopterin:coenzyme M methyltransferase 58.3 76.4 771 {Methanobacterium thermoautotrophicum} MJ0854 694607 693651 N5-methyl-tetrahydromethanopterin:coenzyme M methyltransferase 62.1 77.5 957 {Methanobacterium thermoautotrophicum} MJ0848 697696 697043 N5-methyl-tetrahydromethanopterin:coenzyme M methyltransferase 63.5 77.8 654 {Methanobacterium thermoautotrophicum} MJ0853 694857 694639 N5-methyl-tetrahydromethanopterin:coenzyme M methyltransferase G 51.1 76.6 219 {Methanobacterium thermoautotrophicum} MJ1169 363822 362122 tungsten formylmethanofuran dehydrogenase, subunit A {Methanobacterium 69.4 81.5 1701 thermoautotrophicum} MJ1194 336096 335260 tungsten formylmethanofuran dehydrogenase, subunit B {Methanobacterium 71.1 84.0 837 thermoautotrophicum} MJ1171 361740 360973 tungsten formylmethanofuran dehydrogenase, subunit C {Methanobacterium 52.7 67.7 768 thermoautotrophicum} MJ0658 887575 886886 tungsten formylmethanofuran dehydrogenase, subunit C related protein 35.4 53.4 690 {Methanobacterium thermoautotrophicum} MJ1168 364202 363852 tungsten formylmethanofuran dehydrogenase, subunit D {Methanobacterium 55.2 74.8 351 thermoautotrophicum} MJ1165 366038 365637 tungsten formylmethanofuran dehydrogenase, subunit E {Methanobacterium 38.3 61.1 402 thermoautotrophicum} MJ1166 365484 364567 tungsten formylmethanofuran dehydrogenase, subunit F {Methanobacterium 47.6 67.4 918 thermoautotrophicum} MJ1167 364516 364271 tungsten formylmethanofuran dehydrogenase, subunit G {Methanobacterium 43.1 58.5 246 thermoautotrophicum} Fatty acid and phospholipid metabolism MJ0705 840072 838927 3-hydroxy-3-methylglutaryl coenzyme A reductase {Haloferax volcanii} 49.8 67.3 1146 MJ1546 1612371 1611697 acyl carrier protein synthase {Pyrococcus furiosus} 63.1 78.0 675 MJ0860 688696 689499 bifunctional short chain isoprenyl diphosphate synthase {Methanobacterium 49.5 71.7 804 thermoautotrophicum} MJ1229 299478 300644 biotin carboxylase {Anabaena sp} 58.9 76.2 1167 MJ1212 316229 316786 CDP-diacylglycerol-serine O-phosphatidyltransferase {Bacillus subtilis} 45.5 63.7 558 MJ1504 1661217 1662188 lipopolysaccharide biosynthesis protein (bp1D) {Bordetella pertussis} 44.3 63.1 972 MJ1087 446091 445231 melvalonate kinase {Schizosaccharomyces pombe} 31.5 53.7 861 MJ1549 1610772 1609735 nonspecific lipid-transfer protein {Pyrococcus furiosus} 46.9 66.0 1038 Purines, pyrimidines, nucleosides, and nucleotides 2′-Deoxyribonucleotide metabolism MJ0832 719820 714604 anaerobic ribonucleoside-triphosphate reductase {Escherichia coli} 28.1 49.9 5217 MJ0430 1085497 1086009 deoxycytidine triphosphate deaminase {Desulfurolobus ambivalens} 40.4 61.5 513 MJ1102 429115 428648 deoxycytidine triphosphate deaminase, putative {Desulfurolobus ambivalens} 32.1 53.2 468 MJ0511 1019410 1020075 deoxyuridylate hydroxymethylase {Methanobacterium thermoautotrophicum} 39.4 59.6 666 MJ0937 606252 604921 glycinamide ribonucleotide synthetase {Homo sapiens} 37.1 55.0 1332 Purine ribonucleotide biosynthesis MJ0929 613484 612135 adenylosuccinate lyase {Bacillus subtilis} 42.6 67.4 1350 MJ0561 976592 975741 adenylosuccinate synthetase {Haemophilus influenzae} 41.0 59.1 852 MJ1575 1586386 1585823 GMP synthetase {Borrelia burgdorferi} 41.4 66.7 564 MJ1131 399509 400264 GMP synthetase {Haemophilus influenzae} 52.0 72.3 756 MJ1616 1545605 1544271 inosine-5-monophosphate dehydrogenase {Pyrococcus furiosus} 61.8 80.4 1335 MJ1265 262116 262436 nucleoside diphosphate kinase {Haemophilus influenzae} 51.5 68.3 321 MJ0616 925486 925941 phosphoribosylaminoimidazole carboxylase {Methanobrevibacter smithii} 56.3 76.2 456 MJ1592 1572482 1572009 phosphoribosylaminoimidazolesuccinocarboxamide synthase {Bacillus subtilis} 51.0 69.1 474 MJ0203 1277597 1276734 phosphoribosylformylglycinamidine cyclo-ligase {Bacillus subtilis} 42.7 64.4 864 MJ1648 1507541 1507071 phosphoribosylformylglycinamidine synthase I {Bacillus subtilis} 52.9 71.5 471 MJ1264 262585 264714 phosphoribosylformylglycinamidine synthase II {Bacillus subtilis} 43.3 65.1 2130 MJ1486 13611 14633 phosphoribosylglycinamide formyltransferase 2 {Bacillus subtilis} 61.8 75.9 1023 MJ1366 155580 156431 ribose-phosphate pyrophosphokinase {Haemophilus influenzae} 34.1 55.5 852 Pyrimidine ribonucleotide biosynthesis MJ1581 1581578 1580661 aspartate carbamoyltransferase catalytic chain {Escherichia coli} 50.0 70.7 918 MJ1406 104548 104183 aspartate carbamoyltransferase regulatory chain {Escherichia coli} 39.1 65.1 366 MJ1378 145461 144037 carbamoyl-phosphate synthase, large chain {Bacillus subtilis} 59.7 80.0 1425 MJ1381 143097 141328 carbamoyl-phosphate synthase, pyrimidine-specific, large subunit {Bacillus caldolyticus} 54.7 75.7 1770 MJ1019 523003 522041 carbamoyl-phosphate synthase, small chain {Bacillus subtilis} 49.6 69.1 963 MJ1174 358774 360279 CTP synthase {Haemophilus influenzae} 56.7 74.0 1506 MJ0656 888785 888306 cytidylate kinase {Bacillus subtilis} 31.9 59.5 480 MJ1490 8032 6764 dihydroorotase {Bacillus caldolyticus} 34.5 56.3 1269 MJ0654 889442 890284 dihydroorotase dehydrogenase {Bacillus subtilis} 43.1 66.6 843 MJ0293 1196756 1196196 thymidylate kinase {Schizosaccharomyces pombe} 31.2 58.7 561 MJ1109 421875 421348 uridine 5′-monophosphate synthase {Dictyostelium discoideum} 38.4 64.6 528 MJ1259 271220 270543 uridylate kinase {Haemophilus influenzae} 27.5 48.7 678 Salvage of nucleosides and nucleotides MJ1459 43987 42413 adenine deaminase {Bacillus subtilis} 35.9 61.7 1575 MJ1655 1499440 1499075 adenine phosphoribosyltransferase {Haemophilus influenzae} 35.8 62.5 366 MJ0060 1412894 1412139 methylthioadenosine phosphorylase {Homo sapiens} 41.3 63.2 756 MJ0667 879550 878150 thymidine phosphorylase {Mycoplasma genitalium} 30.5 52.2 1401 Sugar-nucleotide biosynthesis and conversions MJ1101 430386 429235 glucose-1-phosphate thymidylyltransferase {Streptomyces griseus} 32.0 56.0 1152 MJ1334 188314 189084 UDP-glucose pyrophosphorylase {Mycoplasma genitalium} 42.7 63.6 771 Regulatory functions MJ0800 748410 747352 activator of (R)-2-hydroxyglutaryl-CoA dehydratase {Acidaminococcus fermentans} 31.8 51.2 1059 MJ0004 1466944 1466255 activator of (R)-2-hydroxyglutaryl-CoA dehydratase {Acidaminococcus fermentans} 39.0 61.1 690 MJ1344 180975 181229 nitrogen regulatory protein P-II {Haemophilus influenzae} 56.5 73.0 255 MJ0059 1413301 1413047 nitrogen regulatory protein P-II {Haemophilus influenzae} 56.5 75.3 255 MJ0300 1188832 1188194 putative transcriptional regulator {Bacillus subtilis} 27.8 50.3 639 MJ0151 1325766 1325323 putative transcriptional regulator {Pyrococcus furiosus} 51.0 65.0 444 MJ0723 815573 815190 putative transcriptional regulator {Pyrococcus furiosus} 51.2 82.3 384 Replication Degradation of DNA MJ1434 68536 68048 endonuclease III {Bacillus subtilis} 28.7 58.1 489 MJ0613 927393 928424 endonuclease III {Bacillus subtilis} 41.3 66.3 1032 MJ1439 65786 65208 thermonuclease precursor {Staphylococcus hyicus} 36.8 64.1 579 DNA replication, restriction, modification, recombination, and repair MJ1029 510633 509875 dimethyladenosine transferase {Bacillus subtilis} 38.4 58.8 759 MJ0104 1373055 1371130 DNA helicase, putative {Homo sapiens} 35.2 56.7 1926 MJ0171 1297428 1299053 DNA ligase {Desulfurolobus ambivalens} 35.8 62.4 1626 MJ0869 680404 679445 DNA repair protein {Saccharomyces cerevisiae} 44.6 62.2 960 MJ1444 58945 58052 DNA repair protein RAD2 {Homo sapiens} 37.3 63.5 894 MJ0254 1232179 1231757 DNA repair protein RAD51 {Homo sapiens} 32.5 58.4 423 MJ0961 579580 577424 DNA replication initiator protein {Xenopus laevis} 28.1 40.0 2157 MJ1652 1503610 1501559 DNA topoisomerase I {Mycoplasma genitalium} 34.0 55.0 2052 MJ0885 656470 660960 DNA-dependent DNA polymerase family B {Pyrococcus sp.} 47.3 68.0 4491 MJ1529 1630880 1630413 methylated DNA protein cysteine methyltransferase {Haemophilus influenzae} 35.9 66.4 468 MJ1498 1548 715 modification methylase {Haemophilus parainfluenzae} 31.6 52.2 834 MJ0598 942522 941860 modification methylase {Haemophilus influenzae} 32.4 53.8 663 MJ1328 193775 192987 modification methylase {Haemophilus influenzae} 31.1 56.1 789 MJ0563 974521 975309 modification methylase {Methanobacterium thermoformicicum} 34.7 56.2 789 MJ1200 326214 327248 modification methylase {Desulfovibrio desulfuricans} 39.7 56.7 1035 MJ0985 555045 555896 modification methylase {Methanobacterium thermoformicicum} 54.5 73.0 852 MJ1149 383742 384248 mutator mutT protein {Escherichia coli} 40.3 63.9 507 MJ0942 600802 598916 probable ATP dependent helicase {Haemophilus influenzae} 31.9 54.7 1887 MJ0247 1237945 1237322 proliferating-cell nuclear antigen {Saccharomyces cerevisiae} 31.5 54.3 624 MJ0026 1444598 1445224 proliferating-cell nucleolar antigen, 120 kDa {Homo sapiens} 48.1 66.1 627 MJ1422 79304 84727 replication factor C {Homo sapiens} 45.2 64.6 5424 MJ0884 662042 660969 replication factor C, large subunit {Homo sapiens} 32.5 49.2 1074 MJ1220 308420 310102 restriction modification enzyme, subunit M1 {Mycoplasma pulmonis} 32.9 54.4 1683 MJ0132 1345009 1345548 restriction modification enzyme, subunit M1 {Mycoplasma pulmonis} 37.3 61.1 540 MJ0130 1346511 1347179 restriction modification system S subunit {Spiroplasma citri} 29.3 59.2 669 MJ1512 1653580 1648742 reverse gyrase {Sulfolobus acidocaldarius} 41.8 62.4 4839 MJ0135 1341301 1341939 ribonuclease HII (rnhB) {Escherichia coli} 45.2 64.6 639 MJECL42 55944 54271 type I restriction enyzme ECOR124/3 I M protein {Haemophilus influenzae} 39.7 61.4 1673 MJ0124 1349371 1352847 type I restriction enzyme {Haemophilus influenzae} 31.1 52.2 3477 MJ1214 313714 315828 type I restriction enzyme {Haemophilus influenzae} 29.5 52.2 2115 MJECL40 52581 49456 type I restriction enzyme {Haemophilus influenzae} 36.2 59.9 3125 MJ1531 1629137 1628493 type I restriction enzyme CfrI, specificity subunit {Citrobacter freundii} 38.4 57.9 645 MJ1218 310547 311776 type I restriction-modification enzyme, S subunit {Escherichia coli} 29.7 49.7 1230 MJ0984 556397 555909 type II restriction enzyme {Methanobacterium thermoformicicum} 45.9 67.2 489 MJ0600 940932 940315 type II restriction enzyme DPNII {Streptococcus pneumoniae} 46.0 67.4 618 Transcription DNA-dependent RNA polymerases MJ1042 497715 493732 DNA-dependent RNA polymerase, subunit A′ {Methanococcus vannielii} 74.5 88.1 3984 MJ1043 493546 491078 DNA-dependent RNA polymerase, subunit A″ {Methanococcus vannielii} 66.7 83.5 2469 MJ1041 499305 497866 DNA-dependent RNA polymerase, subunit B′ {Methanococcus vannielii} 76.3 91.3 1440 MJ1040 501124 499862 DNA-dependent RNA polymerase, subunit B″ {Methanococcus vannielii} 72.7 87.4 1263 MJ0192 1283621 1283148 DNA-dependent RNA polymerase, subunit D {Arabidopsis thaliana} 39.5 58.6 474 MJ0397 1113901 1114371 DNA-dependent RNA polymerase, subunit E′ {Sulfolobus acidocaldarius} 47.9 70.8 471 MJ0396 1114384 1114560 DNA-dependent RNA polymerase, subunit E″ {Sulfolobus acidocaldarius} 35.9 62.3 177 MJ1039 501599 501366 DNA-dependent RNA polymerase, subunit H {Methanococcus vannielii} 49.4 78.7 234 MJ1390 134111 134350 DNA-dependent RNA polymerase, subunit I {Sulfolobus acidocaldarius} −0.9 −0.9 240 MJ0197 1281417 1281247 DNA-dependent RNA polymerase, subunit K {Haloarcula marismortui} 43.5 65.3 171 MJ0387 1119216 1119512 DNA-dependent PNA polymerase, subunit L {Sulfolobus acidocaldarius} 35.6 63.4 297 MJ0196 1281779 1281561 DNA-dependent RNA polymerase, subunit N {Haloarcula marismortui} 53.8 83.4 219 Transcription factors MJ0941 601867 600923 putative transcription initiation factor IIIC {Saccharomyces cerevisiae} 20.1 44.1 945 MJ1045 490363 489848 putative transcription termination-antitermination factor nusA {Methanococcus vannielii} 47.9 73.7 516 MJ0372 1134509 1134123 putative transcription termination-antitermination factor nusG {Homo sapiens} 38.6 63.8 387 MJ0507 1024170 1024631 TATA-binding transcription initiation factor {Thermococcus celer} 51.4 74.0 462 MJ0782 766586 768592 transcription initiation factor IIB {Pyrococcus woesei} 63.8 77.6 2007 MJ1148 384277 384567 transcription-associated protein, (‘TFIIS’) {Thermococcus celer} 56.4 69.0 291 RNA processing MJ0697 849814 849125 fibrillarin-like pre-rRNA processing protein {Methanococcus vannielii} 75.3 88.3 690 Translation MJ0160 1308036 1309265 PET112 protein {Saccharomyces cerevisiae} 32.3 53.7 1230 Amino acyl tRNA synthetases MJ0564 971657 974149 alanyl-tRNA synthetase (alaRS) {Haemophilus influenzae} 28.0 53.1 2493 MJ0237 1244137 1242641 arginyl-tRNA synthetase {Mycobacterium leprae} 31.3 52.7 1497 MJ1555 1605935 1604679 aspartyl-tRNA synthetase {Pyrococcus sp.} 57.8 75.6 1257 MJ1377 145796 147325 glutamyl-tRNA synthetase {Methanobacterium thermoautotrophicum} 51.7 73.6 1530 MJ0228 1253254 1251524 glycyl tRNA synthetase {Schizosaccharomyces pombe} 45.8 65.2 1731 MJ1000 543634 542396 histidyl-tRNA synthetase {Streptococcus equisimilis} 35.5 56.3 1239 MJ0947 591914 594817 isoleucyl-tRNA synthetase {Methanobacterium thermoautotrophicum} 52.1 70.0 2904 MJ0633 912642 910015 leucyl-tRNA synthetase {Saccharomyces cerevisiae} 34.4 54.9 2628 MJ1263 266697 264745 methionyl-tRNA synthetase {Haemophilus influenzae} 35.6 56.0 1953 MJ0487 1041343 1039994 phenylalanyl-tRNA synthetase, subunit alpha {Saccharomyces cerevisiae} 41.0 64.0 1350 MJ1108 423555 425198 phenylalanyl-tRNA synthetase, subunit beta {Saccharomyces cerevisiae} 31.6 55.4 1644 MJ1238 287985 289172 prolyl-tRNA synthetase {Homo sapiens} 39.3 59.5 1188 MJ1197 332116 330257 threonyl-tRNA synthetase {Synechocystis sp.} 29.1 52.1 1860 MJ1415 96418 95369 tryptophanyl-tRNA synthetase {Schizosaccharomyces pombe} 30.5 55.3 1050 MJ0389 1118380 1117616 tyrosyl-tRNA synthetase {Homo sapiens} 39.9 63.7 765 MJ1007 536642 534186 valyl-tRNA synthetase {Bacillus stearothermophilus} 36.1 56.6 2457 Degradation of proteins, peptides, and glycopeptides MJ1176 356300 357370 ATP-dependent 26S protease regulatory subunit 4 {Homo sapiens} 51.0 74.1 1071 MJ1494 4302 5123 ATP-dependent 26S protease regulatory subunit 8 {Methanobacterium 58.6 78.2 822 thermoautotrophicum} MJ1417 93716 91932 ATP-dependent protease La {Bacillus brevis} 32.8 54.3 1785 MJ0090 1387867 1386755 collagenase {Porphyromonas gingivalis} 32.6 35.2 1113 MJ1130 400455 401969 O-sialoglycoprotein endopeptidase {Saccharomyces cerevisiae} 50.6 67.9 1515 MJ0651 891988 892842 protease IV {Haemophilus influenzae} 35.0 56.2 855 MJ0591 947601 946861 proteasome, subunit alpha {Methanosarcina thermophila} 57.5 78.8 741 MJ1237 289440 289967 proteasome, subunit beta {Methanosarcina thermophila} 47.5 68.2 528 MJ0806 742381 743364 xaa-pro dipeptidase {Lactobacillus delbrueckii} 36.1 65.2 984 MJ0996 547987 546635 Zn protease {Haemophilus influenzae} 33.9 55.0 1353 Protein modification MJ0814 733804 734793 deoxyhypusine synthase {Homo sapiens} 50.0 70.7 990 MJ1274 253925 254653 diphthine synthase {Saccharomyces cerevisiae} 40.7 61.5 729 MJ0172 1296723 1297175 L-isoaspartyl protein carboxyl methyltransferase {Escherichia coli} 47.6 59.4 453 MJ1329 192979 192098 methionine aminopeptidase {Saccharomyces cerevisiae} 36.2 55.1 882 MJ1530 1630123 1629764 N-terminal acetyltransferase complex, subunit ARD1 {Homo sapiens} 39.7 55.7 360 MJ1591 1573833 1573072 selenium donor protein {Homo sapiens} 34.3 57.1 762 Ribosomal proteins: synthesis and modification MJ0509 1022576 1023502 acidic ribisomal protein P0 (L10E) {Methanococcus vannielii} 63.2 82.1 927 MJ0242 1240163 1240228 ribosomal protein HG12 {Catus (cat)} 63.7 81.9 66 MJ1203 325110 325460 ribosomal protein HS6-type {Haloarcula marismortui} 47.0 71.4 351 MJ0510 1021912 1022460 ribosomal protein L1 {Methanococcus vannielii} 64.5 80.3 549 MJ0373 1133926 1133540 ribosomal protein L11 {Sulfolobus solfataricus} 47.2 72.4 387 MJ0508 1023632 1023937 ribosomal protein L12 {Methanococcus vannielii} 72.8 80.9 306 MJ0194 1282568 1282260 ribosomal protein L13 {Haloarcula marismortui} 44.9 66.4 309 MJ0466 1058694 1058452 ribosomal protein L14 {Methanococcus vannielii} 78.8 92.5 243 MJ0657 888216 887977 ribosomal protein L14B {Saccharomyces cerevisiae} 36.4 59.8 240 MJ0477 1052625 1052302 ribosomal protein L15 {Methanococcus vannielii} 62.7 79.5 324 MJ0983 556982 557290 ribosomal protein L15B {Thermoplasma acidophilum} 62.3 78.6 309 MJ0474 1054523 1053939 ribosomal protein L18 {Methanococcus vannielii} 73.3 84.3 585 MJ0473 1054978 1054559 ribosomal protein L19 {Methanococcus vannielii} 67.0 86.4 420 MJ0179 1291786 1291052 ribosomal protein L2 {Methanococcus vannielii} 74.0 87.0 735 MJ0040 1431958 1432260 ribosomal protein L21 {Haloarcula marismortui} 54.5 62.3 303 MJ0460 1061493 1061089 ribosomal protein L22 {Haloarcula marismortui} 40.7 61.7 405 MJ0178 1292097 1291840 ribosomal protein L23 {Methanococcus vannielii} 69.8 91.9 258 MJ0467 1058340 1058062 ribosomal protein L24 {Methanococcus vannielii} 70.5 83.0 279 MJ1201 325929 326078 ribosomal protein L24E {Haloarcula marismortui} 54.6 66.7 150 MJ0462 1060388 1060212 ribosomal protein L29 {Halobacterium halobium} 51.0 69.9 177 MJ0193 1283076 1282705 ribosomal protein L29E {Haloarcula marismortui} 48.7 68.7 372 MJ0176 1293794 1292934 ribosomal protein L3 {Haloarcula marismortui} 45.2 63.9 861 MJ1044 490704 490399 ribosomal protein L30 {Methanococcus vannielii} 63.9 84.1 306 MJ0049 1421907 1422152 ribosomal protein L31{Nicotiana glutinosa} 40.9 66.2 246 MJ0472 1055464 1055063 ribosomal protein L32 {Methanococcus vannielii} 58.0 77.4 402 MJ0655 889197 888931 ribosomal protein L34 {Aedes albopictus} 36.8 58.3 267 MJ0098 1380525 1380686 ribosomal protein L37 {Leishmania infantum,} 50.0 67.4 162 MJ0593 945958 945683 ribosomal protein L37a {Homo sapiens} 44.6 58.7 276 MJ0177 1292889 1292134 ribosomal protein L4 (human) {Haloarcula marismortui} 49.4 66.3 756 MJ0707 838122 838229 ribosomal protein L40 {Saccharomyces cerevisiae} 57.6 66.7 108 MJ0249 1236729 1236448 ribosomal protein L44 {Haloarcula marismortui} 38.8 58.1 282 MJ0689 854995 855150 ribosomal protein L46 {Sulfolobus solfataricus,} 52.0 70.0 156 MJ0469 1057259 1056723 ribosomal protein L5 {Methanococcus vannielii} 72.5 84.5 537 MJ0471 1056071 1055526 ribosomal protein L6 {Methanococcus vannielii} 66.5 82.5 546 MJ0476 1053137 1052745 ribosomal protein L7 {Methanococcus vannielii} 70.3 88.6 393 MJ0595 944670 944473 ribosomal protein LX {Sulfolobus acidocaldarius} 38.9 66.7 198 MJ0322 1172916 1173218 ribosomal protein S10 {Pyrococcus woesei} 67.0 91.0 303 MJ0191 1283956 1283735 ribosomal protein S11 {Haloarcula marismortui} 67.2 80.0 222 MJ1046 489559 489260 ribosomal protein S12 {Methanococcus vannielii} 87.0 96.0 300 MJ0036 1434801 1434352 ribosomal protein S13 {Brugia pahangi,} 49.4 71.0 450 MJ1474 26554 26054 ribosomal protein S15A {Brassica napus} 21.7 48.2 501 MJ0465 1059233 1058883 ribosomal protein S17 {Methanococcus vannielii} 71.6 82.4 351 MJ0245 1238750 1238896 ribosomal protein S17B {Saccharomyces cerevisiae} 55.4 80.9 147 MJ0189 1285220 1284771 ribosomal protein S18 {Arabidopsis thaliana} 42.3 68.5 450 MJ0180 1290861 1290508 ribosomal protein S19 {Haloarcula marismortui} 56.9 73.3 354 MJ0692 853669 854046 ribosomal protein S19S {Ascaris suum} 49.6 67.0 378 MJ0394 1115064 1115366 ribosomal protein S24 {Haloarcula marismortui} 42.6 64.4 303 MJ0250 1236377 1236192 ribosomal protein S27 {Saccharomyces cerevisiae} 42.6 53.8 186 MJ0393 1115369 1115548 ribosomal protein S27A {Caenorhabditis elegans} 58.4 68.8 180 MJ0461 1061060 1060437 ribosomal protein S3 {Haloarcula marismortui} 49.1 72.1 624 MJ1202 325575 325808 ribosomal protein S33 {Kluyveromyces lactis} 62.1 81.1 234 MJ0980 558761 559252 ribosomal protein S3a {Catharanthus roseus} 29.8 52.1 492 MJ0190 1284710 1284150 ribosomal protein S4 {Sulfolobus acidocaldarius} 51.3 68.4 561 MJ0468 1057935 1057318 ribosomal protein S4E {Methanococcus vannielii} 70.9 84.5 618 MJ0475 1053877 1053275 ribosomal protein S5 {Methanococcus vannielii} 75.7 88.6 603 MJ1260 270075 269683 ribosomal protein S6 {Homo sapiens} 36.2 58.0 393 MJ0620 922671 921799 ribosomal protein S6 modification protein {Haemophilus influenzae} 34.4 57.3 873 MJ1001 542227 541487 ribosomal protein S6 modification protein II {Haemophilus influenzae} 24.8 47.4 741 MJ1047 489046 488627 ribosomal protein S7 {Methanococcus vannielii} 65.8 83.6 420 MJ0470 1056445 1056113 ribosomal protein S8 {Methanococcus vannielii} 71.2 89.2 333 MJ0673 873106 872720 ribosomal protein S8E {Haloarcula marismortui} 50.0 69.7 387 MJ0195 1282118 1281840 ribosomal protein S9 {Haloarcula marismortui} 50.0 75.0 279 tRNA modification MJ0946 595006 596040 N2,N2-dimethylguanosine tRNA methyltransferase {Saccharomyces cerevisiae} 31.6 56.0 1035 MJ1675 1478684 1477755 pseudouridylate synthase I {Haemophilus influenzae} 33.5 57.2 930 MJ0436 1081116 1082732 queuine tRNA ribosyltransferase {Escherichia coli} 30.4 47.6 1617 Translation factors MJ0829 723534 722260 peptide chain release factor, eRF, subunit 1 {Xenopus laevis} 33.0 57.3 1275 MJ1505 1659133 1661085 putative ATP-dependent RNA helicase, eIF-4A family {Saccharomyces cerevisiae} 30.8 51.9 1953 MJ1574 1587062 1588927 putative ATP-dependent RNA helicase, eIF-4A family {Bacillus subtilis} 33.1 56.0 1866 MJ0669 876636 877637 putative ATP-dependent RNA helicase, eIF-4A family {Bacillus subtilis} 44.5 65.8 1002 MJ0495 1035432 1034644 putative translation factor, EF-TU/1 alpha family {Thermus aquaticus} 36.9 55.9 1389 MJ0262 1225060 1221653 putative translation initiation factor, FUN12/bIF-2 family {Saccharomyces cerevisiae} 39.3 61.5 3408 MJ0324 1171724 1172830 translation elongation factor, EF-1 alpha {Methanococcus vannielii} 78.9 90.8 1107 MJ1048 488471 486336 translation elongation factor, EF-2 {Methanococcus vannielii} 74.8 88.5 2136 MJ0445 1073262 1073483 translation initiation factor, eIF-1A {Thermoplasma acidophilum} 52.8 70.3 222 MJ0117 1357516 1358196 translation initiation factor, eIF-2, subunit alpha {Saccharomyces cerevisiae} 32.2 56.5 681 MJ0097 1380885 1381313 translation initiation factor, eIF-2, subunit beta {Drosophila melanogaster} 32.1 60.4 429 MJ1261 269396 268164 translation initiation factor, eIF-2, subunit gamma {Homo sapiens} 52.6 71.9 1233 MJ0454 1066217 1067065 translation initiation factor, eIF-2B, subunit alpha {Saccharomyces cerevisiae} 37.9 56.4 849 MJ0122 1353264 1354127 translation initiation factor, eIP-2B, subunit delta {Mus musculus} 29.4 54.6 864 MJ1228 300895 301236 translation initiation factor, eIF-5a {Sulfolobus acidocaldarius} 50.0 69.7 342 Transport and binding proteins MJ0719 818577 820289 ABC transporter ATP-binding protein {Saccharomyces cerevisiae} 49.6 66.9 1713 MJ1023 518606 517821 ABC transporter ATP-binding protein {Bacillus firmus} 49.2 72.4 786 MJ1572 1590114 1589518 ABC transporter ATP-binding protein {Mycoplasma genitalium} 50.0 87.5 597 MJ0035 1435236 1435829 ABC transporter subunit {Cyanelle Cyanophora} 33.9 58.1 594 MJ1508 1656015 1655446 ABC transporter, probable ATP-binding subunit {Haemophilus influenzae} 45.7 68.3 570 MJ1332 189987 191117 GTP-binding protein {Saccharomyces cerevisiae} 38.7 59.8 1131 MJ1326 196392 195292 GTP-binding protein {Schizosaccharomyces pombe} 51.4 71.5 1101 MJ1408 103449 102430 GTP-binding protein, GTP1/OBG-family {Saccharomyces cerevisiae} 30.5 58.4 1020 MJ1464 39865 38858 hypothetical GTP-binding protein (SP:P40010) {Saccharomyces cerevisiae} 32.0 55.5 1008 MJ1033 507274 506324 magnesium and cobalt transport protein {Haemophilus influenzae} 42.2 57.9 951 MJ0091 1386551 1385751 Na+/Ca+ exchanger protein {Escherichia coli} 32.3 58.6 801 MJ0283 1204330 1203563 nucleotide-binding protein {Homo sapiens} 47.5 68.0 768 Amino acids; peptides and amines MJ0609 933328 934587 amino acid transporter {Arabidopsis thaliana} 21.9 48.7 1260 MJ1343 181359 182519 ammonium transport protein AMT1 {Arabidopsis thaliana} 35.6 53.3 1161 MJ0058 1413598 1414770 ammonium transporter {Escherichia coli} 34.2 52.2 1173 MJ1269 258901 257993 branched-chain amino acid transport protein livH {Escherichia coli} 30.8 54.6 909 MJ1266 261404 260577 branched-chain amino acid transport protein livJ {Escherichia coli} 28.8 55.2 828 MJ1270 257896 256934 branched-chain amino acid transport protein livM {Escherichia coli} 28.7 52.2 963 MJ1196 332430 333311 cationic amino acid transporter MCAT-2 {Mus musculus} 24.6 50.6 882 MJ0304 1185908 1186333 ferripyochelin binding protein {Pseudomonas aeruginosa} 55.6 74.7 426 MJ0796 752786 752118 glutamine transport ATP-binding protein Q {Escherichia coli} 47.9 67.2 669 MJ1267 260465 259707 high-affinity branched-chain amino acid transport ATP-binding protein 34.2 60.8 759 {Pseudomonas aeruginosa} MJ1268 259458 258973 high-affinity branched-chain amino acid transport ATP-binding protein 40.4 68.6 486 {Salmonella typhimurium} Anions MJ0412 1099862 1100608 nitrate transport ATP-binding protein {Synechococcus sp} 44.6 70.1 747 MJ0413 1099077 1099826 nitrate transport perinease protein {Synechococcus sp} 34.2 59.4 750 MJ1012 529685 530431 phosphate transport system ATP-binding protein {Escherichia coli} 60.9 80.7 747 MJ1013 528941 529642 phosphate transport system permease protein A {Haemophilus influenzae} 39.6 60.5 702 MJ1014 528397 528810 phosphate transport system permease prdtein C {Haemophilus influenzae} 40.0 66.5 414 MJ1009 532458 533165 phosphate transport system regulatory protein {Escherichia coli} 28.5 54.6 708 MJ1015 526871 527698 phosphate-binding protein {Xanthomonas oryzae} 45.8 60.2 828 Carbohydrates, organic alcohols, and acids MJ0576 960439 959399 malic acid transport protein {Schizosaccharomyces pombe} 23.8 47.9 1041 MJ0762 786703 787524 malic acid transport protein {Schizosaccharomyces pombe} 26.5 49.3 822 MJ0121 1354728 1355291 SN-glycerol-3-phosphate transport ATP-binding protein {Escherichia coli} 33.4 51.7 564 MJ1319 206861 205926 sodium-dependent noradrenaline transporter {Haemophilus influenzae} 37.8 61.0 936 Cations MJ1088 444480 445223 cobalt transport ATP-binding protein O {Salmonella typhimurium} 46.1 66.6 744 MJ1090 443372 443527 cobalt transport protein N {Salmonella typhimurium} 59.1 79.6 156 MJ1089 443778 444374 cobalt transport protein Q {Salmonella typhimurium} 28.9 55.6 597 MJ0089 1388820 1388059 ferric enterobactin transport ATP-binding protein {Escherichia coli} 33.1 59.6 762 MJ0873 674824 674123 ferric enterobactin transport ATP-binding protein {Escherichia coli} 31.5 60.3 702 MJ0566 967842 969857 ferrous iron transport protein B {Escherichia coli} 35.8 61.2 2016 MJ0877 670239 670442 hemin permease {Haemophilus influenzae} 27.9 62.3 204 MJ0087 1390284 1389385 hemin permease {Yersinia enterocolitica} 40.6 67.7 900 MJ0085 1392668 1391613 iron tranport system binding protein {Bacillus subtilis} 32.9 53.3 1056 MJ0876 670677 671498 iron(III) dicitrate transport system permease protein {Escherichia coli} 30.8 52.8 822 MJ1441 64080 60403 magnesium chelatase subunit {Arabidopsis thaliana} 35.3 57.3 3678 MJ0911 628932 629972 magnesium-chelatase subunit {Euglena gracilis} 54.9 73.4 1041 MJ1275 253661 252597 NA(+)/H(+) antiporter {Enterococcus hirae} 29.8 59.9 1065 MJ0672 873748 874665 Na⁺ transporter {Haemophilus influenzae} 39.3 63.1 918 MJ1231 297233 298873 oxaloacetate decarboxylase, alpha subunit {Salmonella typhimurium} 52.0 68.7 1641 MJ1357 164247 165065 putative potassium channel protein {Bacillus cereus} 42.9 66.7 819 MJ1367 154669 155559 sulfate permease (cysA) {Synechococcus sp} 38.5 64.5 891 MJ1368 153995 154666 sulfate/thiosulfate transport protein {Escherichia coli} 30.9 59.4 672 MJ1485 16909 15713 TRK system potassium uptake protein {Escherichia coli} 29.5 58.5 1197 MJ1105 426702 427217 TRK system potassium uptake protein A {Methanosarcina mazei} 39.3 57.6 516 Other MJ1142 390844 389885 arsenical pump-driving ATPase {Escherichia coli} 34.7 55.9 960 MJ0822 727897 729522 ATPase, vanadate-senstive {Methanococcus voltae} 48.1 69.0 1626 MJ0718 820399 821523 chromate resistance protein A {Alcaligenes eutrophus} 27.9 52.4 1125 MJ1226 304219 301988 H⁺-transporting ATPase {Arabidopsis thaliana} 45.1 63.7 2232 MJ1560 1600958 1601974 quinolone resistance norA protein protein {Staphylococcus aureus} 28.8 51.1 1017 Other-categories MJ1365 157333 156458 pheromone shutdown protein {Enterococcus faecalis} 31.2 57.2 876 MJECL24 28069 28845 SOJ protein {Bacillus subtilis} 34.0 62.1 776 Drug and analog sensitivity MJ1538 1621434 1620691 K. lactis toxin sensitivity protein KTI12 {Saccharomyces cerevisiae} 28.4 48.8 744 MJ0102 1375563 1375859 phenylacrylic acid decarboxylase {Saccharomyces cerevisiae} 50.0 74.0 297 Phage-related functions and prophages MJ0630 915023 914598 sodium-dependent phosphate transporter {Cricetulus griseus} 32.6 60.8 426 Transposon-related functions MJ0367 1138754 1138080 integrase {Weeksella zoohelcum} 30.9 54.4 675 MJ0017 1455555 1454946 transposase {Bacillus thuringiensis} 29.5 55.0 610 Other MJ1064 466505 467095 acetyltransferase {Escherichia coli} 47.0 62.4 591 MJ1612 1549430 1548297 BcpC phosphonopyruvate decarboxylase {Streptomyces hygroscopicus} 31.1 48.9 1134 MJ0677 868213 869160 ethylene-inducible protein homolog {Hevea brasiliensis} 68.3 81.0 948 MJ0534 1003199 1002072 flavoprotein {Methanobacterium thermoautotrophicum} 34.6 57.2 1128 MJ0748 797504 798673 flavoprotein {Methanobacterium thermoautotrophicum} 67.0 82.6 1170 MJ0256 1230191 1229760 fom2 phosphonopyruvate decarboxylase {Streptomyces wedmorensis} 36.7 58.5 432 MJ1682 1472535 1473320 heat shock protein X {Haemophilus influenzae} 30.4 55.5 786 MJ0866 682753 682367 HIT protein, member of the HIT-family {Saccharomyces cerevisiae} 39.4 64.8 387 MJ0294 1193529 1195817 large helicase related protein, LHR {Escherichia coli} 31.4 53.6 2289 MJ0010 1460660 1459497 phosphonopyruvate decarboxylase {Streptomyces hygroscopicus} 28.0 47.2 1164 MJ0734 805855 806439 rubrerythrin {Clostridium perfringens} 48.9 69.2 585 MJ0559 978287 977490 surE survival protein {Escherichia coli} 34.7 55.6 798 MJ1100 431754 430489 urease operon protein {Mycobacterium leprae} 33.2 55.0 1266 MJ0543 990687 991100 Wilm's tumor suppressor homolog {Arabidopsis thaliana} 45.6 64.9 414 MJ0765 784011 785549 [6Fe-65] prismane-containing protein {Desulfovibrio desulfuricans} 60.2 72.8 1539 Hypothetical MJ0458 1063165 1062518 hypothetical protein {Sulfolobus acidocaldarius} −0.9 −0.9 648 MJ0483 1047280 1048250 hypothetical protein {Saccharomyces cerevisiae} 27.7 48.7 971 MJ0920 620866 621357 hypothetical protein {Mycoplasma genitalium} 28.3 51.3 492 MJ0443 1074680 1075348 hypothetical protein {Saccharomyces cerevisiae} 27.8 52.8 669 MJ0144 1330246 1330962 hypothetical protein {Methanobacterium thermoautotrophicum} 33.4 58.6 717 MJ0044 1426552 1427241 hypothetical protein (GP:D38561_6) {Streptomyces wedmorensis} 24.1 49.8 690 MJ0868 680710 681000 hypothetical protein (GP:D63999_31) {Synechocystis sp.} 42.2 65.0 291 MJ1502 1662923 1663714 hypothetical protein (GP:D64001_24) {Synechocystis sp.} 36.4 60.1 792 MJ1129 402152 402382 hypothetical protein (GP:D64001_53) {Synechocystis sp.} 37.5 57.9 231 MJ0057 1414899 1416176 hypothetical protein (GP:D64003_36) {Synechocystis sp.} 28.4 53.2 1278 MJ1335 187757 187593 hypothetical protein (GP:D64004_11) {Synechocystis sp.} 46.2 63.5 165 MJ0640 902502 903458 hypothetical protein (GP:D64005_53) {Synechocystis sp.} 33.9 58.8 957 MJ1347 177726 177280 hypothetical protein (GP:D64006_36) {Synechocystis sp.} 32.1 58.6 447 MJ0392 1116428 1115556 hypothetical protein (GP:D64006_95) {Synechocystis sp.} 29.1 54.3 873 MJ0590 950234 948222 hypothetical protein (GP:D64044_18) {Escherichia coli} 30.6 52.6 2013 MJ1178 355642 355956 hypothetical protein (GP:L47709_14) {Bacillus subtilis} 27.1 55.3 315 MJ0438 1080099 1079128 hypothetical protein (GP:L47838_15) {Bacillus subtilis} 29.6 55.8 972 MJ0644 898810 898223 hypothetical protein (GP:M18279_1) {Pseudomonas sp.} 28.3 53.4 588 MJ0828 723763 723668 hypothetical protein (GP:M35130_5) {M71467 M71468} 58.1 87.1 96 MJ1526 1632280 1632810 hypothetical protein (GP:M36534_1) {Methanobrevibacter smithii} 42.6 66.5 531 MJ0888 652964 653473 hypothetical protein (GP:U00011_3) {Mycobacterium leprae} 29.5 51.4 510 MJ0729 809665 809321 hypothetical protein (GP:U18744_1) {Bacillus firmus} 29.4 56.9 345 MJ0787 761402 760077 hypothetical protein (GP:U19363_11) {Methanobacterium 49.9 71.9 1326 thermoautotrophicum} MJ0693 852445 853059 hypothetical protein (GP:U19363_2) {Methanobacterium thermoautotrophicum} 42.8 61.9 615 MJ0489 1039414 1038686 hypothetical protein (GP:U19363_4) {Methanobacterium thermoautotrophicum} 41.3 57.5 729 MJ0446 1072662 1071784 hypothetical protein (GP:U19363_5) {Methanobacterium thermoautotrophicum} 29.8 50.7 879 MJ0076 1400741 1400403 hypothetical protein (GP:U19364_10) {Methanobacterium 25.3 56.1 339 thermoautotrophicum} MJ0034 1435995 1436921 hypothetical protein (GP:UI9364_2) {Methanobacterium thermoautotrophicum} 23.9 49.7 927 MJ1251 277892 277392 hypothetical protein (GP:UI9364_4) {Methanobacterium thermoautotrophicum} 37.8 61.0 501 MJ0927 615224 615694 hypothetical protein (GP:U19364_6) {Methanobacterium thermoautotrophicum} 37.9 57.2 471 MJ0785 763999 762923 hypothetical protein (GP:U19364_8) {Methanobacterium thermoautotrophicum} 57.5 76.6 1077 MJ0746 799630 799935 hypothetical protein (GP:U21086_2) {Methanobacterium thermoautotrophicum} 60.3 76.4 306 MJ1155 378926 380485 hypothetical protein (GP:W28377_114) {Escherichia coli} 40.0 63.7 1560 MJ0653 890904 890359 hypothetical protein (GP:U31567_2) {Methanopyrus kandleri} 42.2 64.8 546 MJ0532 1003608 1004750 hypothetical protein (GP:U32666_1) {Methanosarcina barkeri} 39.3 59.5 1143 MJ0674 872153 871623 hypothetical protein (GP:X83963_2) {Thermococcus litoralis} 58.3 76.7 531 MJ1552 1608984 1608592 hypothetical protein (GP:X85250_3) {Pyrococcus furiosus} 48.5 68.0 393 MJ0709 837195 835996 hypothetical protein (GP:X91006_2) {Pyrococcus sp.} 25.1 50.5 1200 MJ0226 1255943 1255389 hypothetical protein (GP:Z49569_1) {Saccharomyces cerevisiae} 39.0 60.6 555 MJ1476 25468 24851 hypothetical protein (HI0380) {Haemophilus influenzae} 39.7 62.6 618 MJ0441 1076859 1076125 hypothetical protein (HI0902) {Haemophilus influenzae} 29.2 51.1 735 MJ1372 151434 150760 hypothetical protein (HI0920) {Haemophilus influenzae} 46.7 67.5 675 MJ0931 611416 610298 hypothetical protein (MG372) {Mycoplasma genitalium} 34.9 59.9 1119 MJ0861 687240 688532 hypothetical protein (MG423) {Mycoplasma genitalium} 33.9 53.9 1293 MJ1252 277977 278609 hypothetical protein (PIR:B48653) {Lactococcus lactis} 32.5 47.2 633 MJ0279 1206983 1206147 hypothetical protein (PIR:S01072) {Desulfurococcus mobilis} 29.2 53.4 837 MJ0299 1189620 1190600 hypothetical protein (PIR:S11602) {Thermoplasma acidophilum} 62.1 76.6 981 MJ1208 320842 319766 hypothetical protein (PIR:S21569) {Metbanobacterium thermoautotrophicum} 55.4 74.8 1077 MJ1533 1625982 1627727 hypothetical protein (PIR:S28724) {Methanococcus vannielii} 67.3 83.3 1746 MJ0323 1172727 1172257 hypothetical protein (PIR:S38467) {Desulfurococcus mobilis} 60.7 71.7 471 MJ1162 368773 369060 hypothetical protein (PIR:S41581) {Methanothermus fervidus} 48.3 67.9 288 MJ0922 619284 619598 hypothetical protein (PIR:S41583) {Methanothermus fervidus} 48.6 73.4 315 MJ0867 681124 682371 hypothetical protein (PIR:S49379) {Pseudomonas aeruginosa} 28.7 55.2 1248 MJ0047 1423924 1424988 hypothetical protein (PIR:S51413) {Saccharomyces cerevisiae} 26.9 49.9 1065 MJ1236 290570 292111 hypothetical protein (PIR:S51413) {Saccharomyces cerevisiae} 33.9 54.6 1542 MJ0162 1306782 1305562 hypothetical protein (PIR:S51413) {Saccharomyces cerevisiae} 32.4 56.4 1221 MJ0928 614493 614957 hypothetical protein (PIR:S51868) {Saccharomyces cerevisiae} 38.4 61.7 465 MJ1625 1535098 1533113 hypothetical protein (PIR:S52522) {Saccharomyces cerevisiae} 27.6 50.4 1986 MJ0862 686185 687054 hypothetical protein (PIR:S52979) {Erwinia herbicola} 35.5 59.2 870 MJ1432 69872 69453 hypothetical protein (PIR:S53543) {Saccharomyces cerevisiae} 38.5 66.0 420 MJ0710 835912 834914 hypothetical protein (SP:P05409) {Methanococcus thermolithotrophicus} 59.2 79.9 999 MJ0170 1299322 1300185 hypothetical protein (SP:P11666) {Escherichia coli} 30.1 54.8 864 MJ1593 1571988 1571740 hypothetical protein (SP:P12049) {Bacillus subtilis} 40.3 69.6 249 MJ0463 1060127 1059819 hypothetical protein (SP:P14021) {Methanococcus vannielii} 78.5 92.2 309 MJ0464 1059719 1059435 hypothetical protein (SP:P14022) {Methanococcus vannielii} 58.8 79.4 285 MJ0136 1340892 1340105 hypothetical protein (SP:P14027) {Methanococcus vannielii} 63.4 87.8 788 MJ0388 1118696 1119244 hypothetical protein (SP:P15886) {Methanococcus vannielii} 46.9 66.3 549 MJ1225 305183 304425 hypothetical protein (SP:P15889) {Thermofilum pendens} 24.1 53.9 759 MJ1133 398771 397509 hypothetical protein (SP:P22349) {Methanobrevibacter smithii} 45.9 67.4 1263 MJ1273 255725 254676 hypothetical protein (SP:P25125) {Thermus aquaticus} 41.4 60.2 1050 MJ1426 76255 75812 hypothetical protein (SP:P25768) {Methanobacterium ivanovii} 47.3 69.3 444 MJ0549 986782 986360 hypothetical protein (SP:P28910) {Escherichia coli} 33.9 59.3 423 MJ0982 557497 558078 hypothetical protein (SP:P29202) {Haloarcula marismortui} 55.9 75.4 582 MJ0990 552446 552658 hypothetical protein (SP:P31065) {Escherichia coli,} 39.2 62.4 213 MJ0326 1170026 1168809 hypothetical protein (SP:P31466) {Escherichia coli} 45.6 71.7 1218 MJ0812 736053 736679 hypothetical protein (SP:P31473) {Escherichia coli} 25.8 54.3 627 MJ0079 1398567 1399694 hypothetical protein (SP:P31473) {Escherichia coli} 38.0 63.3 1128 MJ1586 1578018 1576645 hypothetical protein (SP:P31806) {Escherichia coli} 32.4 52.1 1434 MJ1124 409920 406336 hypothetical protein (SP:P32639) {Saccharomyces cerevisiae} {Saccharomyces cerevisiae} 26.9 51.5 3585 MJ1081 451124 450726 hypothetical protein (SP:P32698) {Escherichia coli} 38.2 62.8 399 MJ1413 97390 97629 hypothetical protein (SP:P33382) {Listeria monocytogenes} 40.0 60.0 240 MJ1170 362086 361820 hypothetical protein (SP:P33382) {Listeria monocytogenes} 42.2 63.9 267 MJ0051 1419978 1419670 hypothetical protein (SP:P34222) {Saccharomyces cerevisiae} 38.5 55.8 309 MJ1523 1636316 1635945 hypothetical protein (SP:P37002) {Escherichia coli} 43.0 65.0 372 MJ0608 934974 935750 hypothetical protein (SP:P37487) {Bacillus subtilis} 44.3 71.4 777 MJ1661 1493414 1493809 hypothetical protein (SP:P37528) {Bacillus subtilis} 47.0 72.6 396 MJ1582 1580646 1579909 hypothetical protein (SP:P37545) {Bacillus subtilis} 35.4 60.6 738 MJ1375 148221 149408 hypothetical protein (SP:P37555) {Bacillus subtilis} 25.0 48.6 1188 MJ0231 1249786 1250814 hypothetical protein (SP:P37869) {Bacillus subtilis} 40.0 44.0 1029 MJ0882 664582 663910 hypothetical protein (SP:P37872) {Bacillus subtilis} 44.0 68.7 673 MJ0043 1429606 1427252 hypothetical protein (SP:P38423) {Bacillus subtilis} {Bacillus subtilis} 45.5 58.4 2355 MJ0048 1422159 1422842 hypothetical protein (SP:P38619) {Sulfolobus acidocaldarius} 36.6 59.1 684 MJ0989 552670 553011 hypothetical protein (SP:P39164) {Escherichia coli} 29.0 51.8 342 MJ1115 415733 416479 hypothetical protein (SP:P39364) {Eschenchia coli} 27.1 48.3 747 MJ1649 1506277 1507068 hypothetical protein (SP:P39587) {Bacillus subtilis} 28.9 48.5 792 MJ0577 959388 958903 hypothetical protein (SP:P42297) {Bacillus subtilis} 31.6 56.4 486 MJ0531 1004977 1004759 hypothetical protein (SP:P42297) {Bacillus subtilis} 43.3 68.7 219 MJ1247 282030 281677 hypothetical protein (SP:P42404) {Bacillus subtilis} 38.4 60.0 354 MJ0486 1041905 1042681 hypothetical protein (SP:P45476) {Escherichia coli} 30.6 55.7 777 MJ0449 1070080 1069565 hypothetical protein (SP:P46348) {Bacillus subtilis} 31.8 60.7 516 MJ0682 861537 864374 hypothetical protein (SP:P46850) {Escherichia coli} 33.4 53.9 2838 MJ1677 1476726 1476376 hypothetical protein (SP:P46851) {Escherichia coli} 40.3 62.0 351 MJ0588 951068 952243 hypothetical protein GP:L07942_2 {Escherichia coli} 31.1 55.0 1176 MJ0225 1256840 1256121 hypothetical protein GP:U00014_23 {Mycobacterium leprae} 27.4 49.0 720 MJ0134 1342043 1342792 hypothetical protein GP:U00017_21{Mycobacterium leprae} 32.2 52.7 750 MJ0376 1130650 1129136 hypothetical proiein GP:U29579_58 {Escherichia coli} 30.1 51.5 1521 MJ0028 1443023 1443844 hypothetical protein HI1305 {Haemophilus influenzae} 27.0 50.0 822 MJ1136 395844 394486 hypothetical protein Lpg22p (GP:U43281_22) {Saccharomyces cerevisiae} 46.2 63.8 1359 MJ0952 588063 588479 hypothetical protein PIR:S49633 {Saccharomyces cerevisiae} 26.8 55.0 417 MJ0403 1109067 1108276 hypothetical protein PIR:S55196 {Saccharomyces cerevisiae} 27.6 48.2 792 MJ1031 509420 508506 hypothetical protein SP:P45869 {Bacillus subtilis} 26.8 51.1 915

TABLE 1B MJ0479 1,050,508 1,049,948 adenylate 100.0% 100.0% 585 kinase {Meth- anococcus jannaschii}

TABLE 2 MJ0002 4071 3343 MJ0003 4911 5378 MJ0008 10075 10734 MJ0009 10743 11570 MJ0011 12983 13459 MJ0012 13927 13427 MJ0013 14836 14351 MJ0014 15455 14820 MJ0015 15514 15804 MJ0016 16416 15866 MJ0018 17658 19229 MJ0019 21121 19232 MJ0021 22762 23886 MJ0023 25284 25637 MJ0024 26105 25689 MJ0025 27122 26109 MJ0027 28572 28021 MJ0037 38073 38786 MJ0038 39443 38793 MJ0039 39974 39654 MJ0041 41838 40477 MJ0042 42527 41883 MJ0045 46506 45907 MJ0046 47351 46569 MJ0050 52237 51050 MJ0052 53374 52709 MJ0053 54068 53388 MJ0054 55001 54159 MJ0056 56154 55759 MJ0062 60618 61238 MJ0063 61322 61855 MJ0064 61897 62454 MJ0065 63551 62463 MJ0066 65078 63657 MJ0067 65160 65468 MJ0068 65861 65517 MJ0070 66966 67211 MJ0071 67211 67480 MJ0072 67562 67693 MJ0073 67729 68007 MJ0074 69089 68016 MJ0075 70324 69236 MJ0077 71539 70394 MJ0078 72674 72054 MJ0080 74182 73802 MJ0086 80788 81903 MJ0088 83019 83537 MJ0093 88517 88092 MJ0094 89481 88564 MJ0095 89828 89568 MJ0096 90752 89967 MJ0100 94823 93297 MJ0103 97958 99256 MJ0105 101649 101239 MJ0106 102541 101840 MJ0107 102733 104295 MJ0109 106419 105664 MJ0110 106880 106614 MJ0114 111874 112782 MJ0115 113249 112785 MJ0116 113931 113257 MJ0119 116397 115726 MJ0120 117070 116372 MJ0123 119524 119195 MJ0125 123378 123031 MJ0126 123685 123392 MJ0127 124034 123672 MJ0128 124341 124048 MJ0129 124487 124996 MJ0131 126783 126475 MJ0133 129427 128609 MJ0137 134976 134119 MJ0138 136566 135121 MJ0139 136616 138244 MJ0140 139150 139539 MJ0141 139529 139825 MJ0142 139797 140237 MJ0145 142991 142188 MJ0146 143409 143203 MJ0147 144813 143701 MJ0149 146003 145830 MJ0150 146069 146587 MJ0154 152143 152589 MJ0157 159807 160085 MJ0158 160155 161276 MJ0159 163046 161430 MJ0163 167378 166818 MJ0164 168614 167430 MJ0165 169394 168627 MJ0166 170194 169430 MJ0173 175871 176341 MJ0175 178089 177475 MJ0181 182625 181918 MJ0182 183311 182730 MJ0183 183491 183348 MJ0184 183606 183827 MJ0185 183886 184032 MJ0187 185874 185440 MJ0188 186674 185880 MJ0198 191384 192259 MJ0201 193486 193007 MJ0202 193687 194454 MJ0206 198871 198467 MJ0207 198967 199419 MJ0208 200166 199429 MJ0209 200956 200159 MJ0212 203759 204019 MJ0213 204137 204583 MJ0215 205636 205190 MJ0223 214474 214163 MJ0224 215072 214566 MJ0227 218176 219099 MJ0229 221136 220852 MJ0230 221386 221144 MJ0233 224281 225111 MJ0235 226124 226369 MJ0236 226362 227639 MJ0239 230506 230988 MJ0240 231618 231094 MJ0241 232062 231628 MJ0243 232563 232318 MJ0248 235142 235651 MJ0251 238728 238288 MJ0252 238849 239487 MJ0255 241359 240607 MJ0257 242764 243696 MJ0258 245039 243840 MJ0259 245717 245112 MJ0261 247082 246423 MJ0263 251686 250727 MJ0270 256421 256188 MJ0271 256902 257441 MJ0272 257452 257649 MJ0273 258107 258412 MJ0274 260378 258819 MJ0275 261121 260516 MJ0280 266375 266758 MJ0281 267291 266761 MJ0282 267341 267787 MJ0284 269902 269174 MJ0286 270849 270499 MJ0287 271160 270870 MJ0288 271755 271222 MJ0289 272805 271801 MJ0290 273753 273121 MJ0292 275409 275137 MJ0296 279767 280360 MJ0297 281155 280406 MJ0298 281290 281739 MJ0301 285101 284220 MJ0303 285971 285558 MJ0305 286594 287778 MJ0306 287997 287818 MJ0308 289084 288386 MJ0310 290609 290268 MJ0311 290981 290652 MJ0312 291845 291228 MJ0314 293767 294369 MJ0315 294826 294455 MJ0316 295458 294964 MJ0317 296374 295733 MJ0319 297675 297902 MJ0320 298001 298645 MJ0321 298675 299040 MJ0325 302095 301172 MJ0327 303625 303927 MJ0328 304755 304318 MJ0329 306607 304760 MJ0330 308266 306620 MJ0331 308670 308266 MJ0332 308995 308678 MJ0333 309670 309410 MJ0334 309816 310112 MJ0335 310179 310919 MJ0336 310932 311288 MJ0337 311299 312084 MJ0338 312100 312402 MJ0339 312374 312694 MJ0340 312697 313398 MJ0341 313411 313770 MJ0342 313918 314286 MJ0343 314270 316807 MJ0344 316820 317359 MJ0345 317314 318264 MJ0346 318277 318579 MJ0347 318593 319045 MJ0348 319620 321995 MJ0349 322367 322053 MJ0350 322681 322418 MJ0351 323154 322705 MJ0352 323901 323185 MJ0353 324142 323891 MJ0354 324296 324123 MJ0355 324661 324374 MJ0356 324957 324697 MJ0357 326407 325943 MJ0358 326796 326413 MJ0359 327449 326808 MJ0360 328174 327770 MJ0361 329502 329182 MJ0362 329659 329847 MJ0364 332163 332495 MJ0365 332503 333030 MJ0366 333033 333308 MJ0368 334581 334886 MJ0369 336040 334934 MJ0371 337418 337639 MJ0374 339873 338884 MJ0375 339920 340681 MJ0377 343243 343752 MJ0378 343921 344886 MJ0379 345500 344889 MJ0380 345657 345974 MJ0381 345977 346936 MJ0382 346955 347683 MJ0383 347677 349518 MJ0384 349546 350259 MJ0385 350252 351304 MJ0386 351648 351307 MJ0390 355149 354760 MJ0395 357787 357314 MJ0398 359111 359923 MJ0400 361593 362411 MJ0401 362717 362520 MJ0402 363046 362729 MJ0404 364804 364355 MJ0405 365385 365002 MJ0408 367518 367880 MJ0409 367946 370054 MJ0410 370074 370865 MJ0414 374603 373419 MJ0415 374712 375197 MJ0416 375222 375791 MJ0417 376510 375800 MJ0418 376627 377388 MJ0419 377369 378430 MJ0420 378394 379533 MJ0421 379640 380719 MJ0423 381855 382031 MJ0424 382046 382336 MJ0425 382317 382712 MJ0426 383243 382704 MJ0427 383719 383243 MJ0431 387350 387135 MJ0432 388127 387852 MJ0433 388663 388139 MJ0434 389342 388677 MJ0435 389620 389342 MJ0437 391903 391667 MJ0439 394280 393234 MJ0440 394492 395292 MJ0444 398609 397740 MJ0447 401037 400555 MJ0448 401168 401935 MJ0450 403277 403834 MJ0452 404962 404519 MJ0453 405287 404967 MJ0455 406863 406285 MJ0456 406888 407943 MJ0459 410088 410354 MJ0480 422470 423063 MJ0481 423792 424085 MJ0482 423793 423074 MJ0485 427056 428102 MJ0488 432390 432854 MJ0491 434681 435106 MJ0492 435385 435101 MJ0494 436499 436891 MJ0496 438482 438823 MJ0497 439219 438821 MJ0498 439679 439212 MJ0500 442304 441537 MJ0501 442990 442394 MJ0504 445785 446372 MJ0505 446365 447117 MJ0512 453993 453292 MJ0513 454868 454149 MJ0517 459731 459321 MJ0518 460018 459737 MJ0519 460275 460033 MJ0521 461746 461549 MJ0522 462422 461769 MJ0523 463226 462534 MJ0524 463697 463239 MJ0525 463997 463839 MJ0526 464308 464123 MJ0527 465146 464655 MJ0528 465442 465149 MJ0529 466215 465520 MJ0538 474805 474026 MJ0539 476422 474833 MJ0540 476947 476693 MJ0541 477507 476971 MJ0545 483451 482711 MJ0546 483623 483456 MJ0548 485032 484589 MJ0550 487106 486012 MJ0551 487918 487106 MJ0553 489383 488925 MJ0554 490365 489910 MJ0556 492396 491875 MJ0557 493186 492572 MJ0558 493984 493202 MJ0560 495301 494891 MJ0562 496903 496691 MJ0565 502486 502046 MJ0567 504742 504497 MJ0568 504847 505221 MJ0570 506837 506112 MJ0572 509860 510117 MJ0573 510262 510828 MJ0574 510865 511143 MJ0575 511121 511807 MJ0580 515428 515075 MJ0581 515692 515937 MJ0582 515940 516323 MJ0583 516393 516563 MJ0584 516563 517657 MJ0585 517680 518294 MJ0586 518563 519057 MJ0587 519994 519536 MJ0589 521451 521768 MJ0592 525620 526357 MJ0594 526886 527392 MJ0596 528074 528475 MJ0597 528539 529612 MJ0599 530524 531120 MJ0602 533752 532970 MJ0604 535443 535144 MJ0605 535634 535443 MJ0606 536194 535922 MJ0607 536435 536199 MJ0610 540394 539093 MJ0614 545444 545061 MJ0618 547877 547584 MJ0619 549378 547861 MJ0621 551088 550573 MJ0623 552787 553362 MJ0625 553606 554613 MJ0626 554709 555335 MJ0627 555369 555719 MJ0628 555715 556203 MJ0629 556208 556849 MJ0632 558292 559380 MJ0634 562682 564565 MJ0635 564797 565636 MJ0638 568586 567912 MJ0639 568870 568586 MJ0642 571462 572451 MJ0645 574498 574743 MJ0646 574757 575248 MJ0647 575457 575296 MJ0648 575881 575441 MJ0650 577458 579521 MJ0652 580869 580471 MJ0659 585626 586039 MJ0660 586366 586136 MJ0661 587014 586496 MJ0662 587657 587007 MJ0664 589291 590163 MJ0665 590629 590180 MJ0668 594556 594314 MJ0670 596945 595887 MJ0675 601925 600753 MJ0678 605240 604263 MJ0683 611696 610920 MJ0686 615407 613668 MJ0687 616482 615478 MJ0688 616670 617110 MJ0690 617965 617375 MJ0691 618300 617974 MJ0694 620244 621365 MJ0695 621809 621486 MJ0696 622409 621933 MJ0699 625837 624698 MJ0700 625851 626822 MJ0701 626831 628063 MJ0702 628050 629831 MJ0703 629859 630536 MJ0704 631069 632199 MJ0706 633440 634081 MJ0708 634868 634425 MJ0711 643995 644960 MJ0712 645967 644963 MJ0714 648530 648880 MJ0716 650013 650270 MJ0717 650815 650459 MJ0724 657809 657189 MJ0730 663605 663048 MJ0731 664213 663620 MJ0733 665883 665521 MJ0737 667834 667652 MJ0738 668149 667877 MJ0739 668627 668175 MJ0742 669819 669496 MJ0745 672208 671675 MJ0747 673416 672961 MJ0749 675903 675151 MJ0750 676710 675997 MJ0751 677628 676795 MJ0752 677942 677715 MJ0753 678766 678146 MJ0754 679347 678775 MJ0755 680644 679619 MJ0756 681296 680889 MJ0757 682155 681424 MJ0758 682653 682213 MJ0759 683029 682700 MJ0760 683871 683047 MJ0761 684833 684072 MJ0763 686251 685889 MJ0764 686611 686264 MJ0766 688821 688729 MJ0767 689531 689100 MJ0768 689589 690335 MJ0769 690987 690481 MJ0770 691651 690983 MJ0772 692429 693487 MJ0773 694540 694016 MJ0774 695228 696454 MJ0775 696438 697379 MJ0776 697375 698523 MJ0777 698474 699046 MJ0778 699097 699603 MJ0779 700509 699613 MJ0780 701537 700533 MJ0783 706171 706737 MJ0786 710078 710620 MJ0788 712303 712539 MJ0789 712625 712972 MJ0790 713001 713696 MJ0792 715511 715777 MJ0793 716398 716931 MJ0794 716992 717405 MJ0795 717488 718999 MJ0797 720647 721759 MJ0798 721779 722780 MJ0799 722786 723667 MJ0801 725037 726173 MJ0802 726398 726961 MJ0803 726984 727499 MJ0804 727530 728387 MJ0805 728332 728994 MJ0807 730149 730670 MJ0808 730806 731804 MJ0809 733025 733525 MJ0810 733584 734255 MJ0811 735675 734359 MJ0815 739584 738697 MJ0816 740542 739652 MJ0817 741119 740502 MJ0818 741733 741125 MJ0819 742225 741899 MJ0820 742295 742191 MJ0821 742765 742598 MJ0823 744830 745600 MJ0826 747462 747875 MJ0830 750568 750101 MJ0831 750950 752245 MJ0833 758976 758239 MJ0834 759796 759083 MJ0835 760901 759822 MJ0836 762786 762430 MJ0837 762860 763606 MJ0838 764466 764816 MJ0839 765906 764857 MJ0840 765992 766972 MJ0841 768225 766981 MJ0856 780538 779996 MJ0857 781920 781099 MJ0858 782318 781980 MJ0859 782837 782355 MJ0865 788311 789585 MJ0871 795055 795975 MJ0872 797236 796022 MJ0874 798213 798491 MJ0875 798611 800854 MJ0878 803147 804388 MJ0880 805402 806325 MJ0883 808397 809404 MJ0887 818880 818209 MJ0889 819606 821000 MJ0890 821429 821019 MJ0894 824064 824486 MJ0895 824467 825492 MJ0896 825552 825953 MJ0897 825946 826362 MJ0898 826495 826932 MJ0899 826954 827643 MJ0900 827668 829308 MJ0901 829430 830998 MJ0902 831028 831729 MJ0903 831942 833855 MJ0904 834299 834547 MJ0905 834622 834954 MJ0906 834959 836056 MJ0907 836917 836072 MJ0909 840933 841220 MJ0910 841954 841433 MJ0912 843688 844416 MJ0914 845908 845783 MJ0915 847507 846707 MJ0916 847875 847609 MJ0917 847950 849671 MJ0919 850996 850550 MJ0921 852470 851571 MJ0923 853368 854258 MJ0925 855529 855212 MJ0926 856378 856638 MJ0933 862692 863390 MJ0935 864824 865447 MJ0936 865545 866042 MJ0938 868207 867473 MJ0939 868278 869102 MJ0943 875111 873870 MJ0944 875300 875659 MJ0945 876358 875687 MJ0948 881231 880668 MJ0949 881637 881269 MJ0950 882370 881684 MJ0951 883634 882570 MJ0953 884488 884787 MJ0954 886106 884802 MJ0956 887437 888216 MJ0957 888219 889268 MJ0958 889276 890553 MJ0962 894937 895320 MJ0966 899875 901197 MJ0967 901940 901326 MJ0968 901996 902814 MJ0969 903935 903126 MJ0970 904627 904199 MJ0971 904756 905844 MJ0972 905808 906488 MJ0973 907728 906496 MJ0974 908172 907741 MJ0975 908365 908162 MJ0976 908463 909560 MJ0977 909594 911000 MJ0978 911359 911688 MJ0979 912309 911719 MJ0981 914246 913641 MJ0986 917606 917373 MJ0987 917909 918247 MJ0988 918361 919347 MJ0991 920189 920608 MJ0992 920924 921142 MJ0995 924316 923636 MJ0997 925109 925719 MJ0998 926425 926012 MJ1002 930965 931891 MJ1004 933349 933990 MJ1005 933994 934386 MJ1006 934412 935437 MJ1010 941079 939958 MJ1011 941860 941471 MJ1016 946060 946941 MJ1017 946934 947542 MJ1020 950418 951194 MJ1021 951732 951244 MJ1022 953674 951968 MJ1024 954536 955744 MJ1025 956917 955751 MJ1028 959569 961611 MJ1030 962492 962932 MJ1032 963985 965082 MJ1034 966050 966310 MJ1036 967587 968276 MJ1049 986885 987367 MJ1050 987438 987968 MJ1052 989793 989503 MJ1053 990349 989861 MJ1060 1000457 1002067 MJ1067 1008238 1008681 MJ1069 1010805 1009630 MJ1070 1011399 1010929 MJ1071 1012337 1011399 MJ1072 1012709 1012362 MJ1073 1013688 1012879 MJ1074 1014135 1013800 MJ1076 1016646 1015636 MJ1077 1018245 1016683 MJ1078 1019039 1018338 MJ1079 1020506 1019316 MJ1080 1021091 1020687 MJ1082 1021657 1022016 MJ1083 1022089 1022667 MJ1085 1023633 1025159 MJ1086 1025159 1026178 MJ1092 1030102 1030743 MJ1094 1033051 1031897 MJ1095 1034350 1033088 MJ1098 1039265 1038627 MJ1099 1040323 1039619 MJ1103 1043990 1043727 MJ1106 1046606 1046052 MJ1107 1047073 1046627 MJ1110 1052574 1051117 MJ1111 1053691 1052540 MJ1112 1053818 1053645 MJ1114 1055795 1055220 MJ1117 1058450 1059037 MJ1118 1059065 1059331 MJ1120 1060339 1061175 MJ1121 1061532 1061251 MJ1122 1061729 1061508 MJ1123 1061809 1062423 MJ1125 1066578 1066399 MJ1126 1067325 1068140 MJ1127 1068204 1069043 MJ1128 1069964 1069050 MJ1132 1073401 1073048 MJ1134 1075567 1074881 MJ1137 1078625 1078035 MJ1138 1078694 1079215 MJ1139 1080031 1079336 MJ1140 1080732 1080049 MJ1141 1080810 1081406 MJ1143 1082498 1083604 MJ1144 1084575 1083607 MJ1145 1085112 1084918 MJ1147 1086431 1087786 MJ1150 1088688 1089230 MJ1151 1089352 1089681 MJ1152 1089693 1089902 MJ1153 1089902 1090087 MJ1154 1091598 1090246 MJ1157 1097614 1098636 MJ1158 1097631 1097245 MJ1159 1098676 1100610 MJ1161 1102129 1102629 MJ1163 1104052 1104747 MJ1164 1106045 1105095 MJ1172 1111539 1111781 MJ1173 1111785 1112066 MJ1177 1117451 1118467 MJ1179 1118839 1119285 MJ1180 1119545 1119979 MJ1181 1120081 1120677 MJ1182 1121087 1122184 MJ1183 1122200 1122670 MJ1184 1122741 1123160 MJ1185 1125032 1123167 MJ1186 1125194 1126231 MJ1188 1127047 1126238 MJ1189 1128908 1128060 MJ1198 1142323 1144605 MJ1199 1145059 1144631 MJ1205 1148679 1148371 MJ1206 1149937 1148675 MJ1207 1150577 1151254 MJ1209 1154047 1152613 MJ1210 1154918 1154148 MJ1211 1155290 1154943 MJ1213 1156520 1156191 MJ1215 1159884 1159639 MJ1216 1160233 1159871 MJ1217 1160540 1160247 MJ1219 1162177 1161875 MJ1221 1164080 1164958 MJ1222 1165703 1164984 MJ1223 1165956 1165681 MJ1224 1167016 1166600 MJ1230 1173450 1173235 MJ1232 1176334 1175447 MJ1233 1176475 1177311 MJ1234 1178669 1177947 MJ1239 1184644 1185318 MJ1240 1185617 1185327 MJ1241 1185877 1185644 MJ1243 1187992 1187624 MJ1244 1188410 1188087 MJ1245 1188760 1188425 MJ1248 1191184 1190723 MJ1249 1191367 1192449 MJ1250 1192973 1193731 MJ1254 1197164 1197400 MJ1255 1197430 1198611 MJ1256 1198911 1199543 MJ1257 1199543 1200589 MJ1262 1204364 1205530 MJ1272 1216145 1216633 MJ1278 1223720 1223184 MJ1279 1224266 1223724 MJ1280 1224460 1224930 MJ1281 1224854 1227994 MJ1282 1228714 1229769 MJ1283 1231676 1231017 MJ1284 1232029 1231667 MJ1285 1232580 1232029 MJ1286 1234269 1232587 MJ1287 1235086 1234319 MJ1288 1235901 1235155 MJ1289 1236778 1236284 MJ1290 1237713 1236778 MJ1291 1238448 1237729 MJ1292 1238662 1241124 MJ1293 1241174 1241866 MJ1295 1243251 1242847 MJ1301 1250120 1248921 MJ1302 1250541 1250149 MJ1304 1252617 1252162 MJ1305 1253036 1252596 MJ1306 1253300 1253052 MJ1307 1254110 1253325 MJ1308 1254426 1254115 MJ1309 1255877 1254459 MJ1310 1256325 1255942 MJ1311 1256457 1257287 MJ1312 1257321 1258283 MJ1313 1258388 1259596 MJ1315 1260519 1261589 MJ1316 1261606 1261833 MJ1317 1263015 1261822 MJ1318 1264868 1263063 MJ1320 1268194 1267802 MJ1321 1270356 1268218 MJ1322 1273392 1270378 MJ1323 1274489 1273392 MJ1325 1275428 1275694 MJ1327 1277081 1277815 MJ1330 1280424 1280792 MJ1331 1281220 1280801 MJ1333 1282515 1282766 MJ1336 1284800 1285282 MJ1337 1285743 1286216 MJ1339 1287389 1287850 MJ1340 1287925 1288266 MJ1341 1289221 1288286 MJ1342 1289457 1289798 MJ1345 1291918 1292841 MJ1348 1295149 1296126 MJ1350 1298227 1297454 MJ1354 1304338 1304772 MJ1355 1304858 1306531 MJ1356 1306729 1307295 MJ1358 1309040 1308648 MJ1359 1309889 1309164 MJ1360 1310249 1309953 MJ1361 1310355 1311230 MJ1364 1313354 1314619 MJ1369 1318564 1319028 MJ1370 1319061 1320044 MJ1371 1320053 1320775 MJ1373 1321601 1322086 MJ1374 1322262 1322954 MJ1379 1328524 1328823 MJ1380 1328819 1329052 MJ1382 1331473 1331036 MJ1383 1332364 1331597 MJ1384 1333177 1332596 MJ1385 1333741 1333205 MJ1386 1333877 1334008 MJ1387 1335433 1334297 MJ1389 1337813 1337412 MJ1393 1341979 1343802 MJ1394 1343895 1346852 MJ1395 1347176 1347571 MJ1396 1347707 1356388 MJ1397 1356457 1357905 MJ1398 1358183 1359355 MJ1399 1359929 1359339 MJ1400 1360142 1359942 MJ1401 1360259 1362682 MJ1402 1364357 1363320 MJ1403 1365794 1364673 MJ1404 1366111 1367364 MJ1405 1367427 1367639 MJ1407 1368408 1368794 MJ1409 1370733 1369939 MJ1410 1371310 1370834 MJ1412 1373210 1374703 MJ1414 1375807 1375094 MJ1416 1378350 1376995 MJ1419 1382016 1381714 MJ1423 1394263 1393208 MJ1424 1394481 1395002 MJ1427 1396680 1397633 MJ1428 1397643 1399343 MJ1429 1399343 1400842 MJ1431 1401322 1402398 MJ1433 1402914 1403654 MJ1435 1404402 1404614 MJ1436 1404758 1405048 MJ1437 1405055 1405738 MJ1440 1407288 1408133 MJ1442 1412130 1412735 MJ1443 1412784 1413104 MJ1445 1414331 1414858 MJ1447 1415840 1416982 MJ1448 1416982 1418571 MJ1449 1418577 1419686 MJ1450 1419699 1420811 MJ1451 1420869 1422320 MJ1452 1422616 1423392 MJ1453 1423398 1423973 MJ1455 1425643 1424729 MJ1457 1427021 1427422 MJ1458 1427487 1428140 MJ1460 1430419 1429943 MJ1461 1431156 1430560 MJ1462 1431506 1431258 MJ1463 1432201 1431530 MJ1466 1436397 1435756 MJ1467 1436562 1437008 MJ1468 1437029 1440055 MJ1469 1440055 1440279 MJ1470 1440747 1442618 MJ1471 1442618 1443151 MJ1472 1443165 1444796 MJ1475 1446447 1446821 MJ1477 1447530 1448537 MJ1478 1449448 1448540 MJ1480 1451452 1452720 MJ1481 1452735 1453373 MJ1483 1454337 1454783 MJ1484 1454768 1455217 MJ1487 1459016 1460293 MJ1488 1460315 1461493 MJ1491 1465684 1466055 MJ1492 1466067 1466534 MJ1493 1466552 1467235 MJ1495 1468532 1469377 MJ1496 1469370 1469711 MJ1497 1469711 1470748 MJ1499 1472128 1471649 MJ1500 1472920 1472363 MJ1501 1473615 1472947 MJ1503 1474982 1474587 MJ1506 1479963 1478767 MJ1507 1480030 1481214 MJ1509 1482024 1482482 MJ1510 1483084 1482506 MJ1511 1483234 1483572 MJ1513 1489601 1488606 MJ1514 1489692 1490078 MJ1515 1490084 1491148 MJ1516 1491173 1491466 MJ1517 1492030 1492863 MJ1518 1492917 1493975 MJ1519 1494094 1497618 MJ1520 1498588 1497656 MJ1521 1498905 1500170 MJ1524 1501404 1501727 MJ1525 1501702 1504500 MJ1527 1505607 1505281 MJ1535 1512870 1513766 MJ1537 1515742 1514714 MJ1539 1516728 1517042 MJ1540 1517209 1517466 MJ1542 1521169 1518746 MJ1544 1523759 1522470 MJ1545 1523900 1524592 MJ1547 1525820 1526005 MJ1548 1526062 1526427 MJ1550 1527849 1528031 MJ1551 1528046 1528216 MJ1553 1528749 1529240 MJ1554 1529326 1531191 MJ1556 1532701 1533636 MJ1557 1533644 1534390 MJ1558 1534666 1534397 MJ1559 1534699 1535262 MJ1561 1538168 1536510 MJ1562 1539331 1538168 MJ1563 1539812 1539345 MJ1564 1540186 1540695 MJ1565 1540699 1542237 MJ1566 1543572 1542232 MJ1567 1544072 1543557 MJ1568 1544632 1544078 MJ1570 1545637 1545981 MJ1571 1546111 1546986 MJ1573 1548452 1548270 MJ1576 1551559 1552164 MJ1577 1552197 1553990 MJ1579 1555146 1554937 MJ1580 1555498 1555127 MJ1583 1557431 1557808 MJ1584 1558268 1557816 MJ1585 1559172 1558255 MJ1587 1560732 1561265 MJ1588 1561285 1561620 MJ1589 1561657 1562379 MJ1590 1562770 1563084 MJ1595 1567357 1566332 MJ1598 1572075 1571026 MJ1599 1572924 1572094 MJ1600 1573002 1573532 MJ1601 1573539 1574018 MJ1604 1578693 1577308 MJ1608 1582917 1583126 MJ1609 1583168 1584289 MJ1613 1589822 1589058 MJ1614 1590582 1589830 MJ1615 1591350 1590586 MJ1617 1593103 1593381 MJ1618 1593786 1593397 MJ1620 1594531 1596084 MJ1621 1596297 1596127 MJ1622 1597169 1597719 MJ1623 1597939 1599474 MJ1624 1599991 1599602 MJ1626 1602381 1600087 MJ1627 1604683 1604231 MJ1628 1606127 1604784 MJ1629 1607293 1606418 MJ1630 1610737 1607330 MJ1631 1611184 1612740 MJ1632 1612697 1613446 MJ1633 1614897 1613467 MJ1634 1615733 1615011 MJ1635 1615933 1617174 MJ1637 1618268 1619686 MJ1638 1620457 1619678 MJ1639 1620605 1621036 MJ1640 1621671 1621057 MJ1641 1622664 1621804 MJ1642 1623032 1623514 MJ1644 1627146 1627667 MJ1646 1628442 1629074 MJ1650 1632586 1631435 MJ1651 1633407 1632631 MJ1653 1635797 1636951 MJ1654 1637097 1637693 MJ1657 1639687 1640427 MJ1658 1640511 1640783 MJ1659 1640800 1641870 MJ1660 1641857 1643503 MJ1664 1646502 1647179 MJ1665 1648555 1647182 MJ1666 1650080 1648686 MJ1667 1651336 1650083 MJ1668 1652321 1651194 MJ1669 1653119 1652376 MJ1670 1653547 1653149 MJ1671 1653684 1653550 MJ1672 1656206 1653807 MJ1673 1656630 1656244 MJ1674 1658539 1656638 MJ1676 1659621 1660334 MJ1678 1660939 1662126 MJ1679 1662142 1662432 MJ1680 1662411 1662866 MJ1681 1663887 1662862 MJECS01 1268 432 MJECS02 4814 1272 MJECS03 5192 4851 MJECS04 5884 5459 MJECS05 6365 6814 MJECS06 7443 7009 MJECS07 8765 7428 MJECS08 11950 8738 MJECS09 12641 11925 MJECS10 14062 13181 MJECS11 14404 15030 MJECS12 16547 15411 MJECL01 275 1048 MJECL02 1474 1085 MJECL03 1700 1377 MJECL04 1865 3250 MJECL05 3235 3450 MJECL06 4170 3787 MJECL07 5844 4561 MJECL08 7415 5832 MJECL09 7780 8103 MJECL10 8107 8784 MJECL11 8788 9159 MJECL12 9150 9887 MJECL13 10678 12483 MJECL14 14468 15427 MJECL15 15420 16541 MJECL16 16599 16811 MJECL18 20873 21505 MJECL19 21456 22019 MJECL20 22829 23290 MJECL21 24596 23298 MJECL22 25120 24854 MJECL23 27628 25136 MJECL25 28835 29167 MJECL26 30215 29178 MJECL27 31077 30571 MJECL28 35352 31534 MJECL30 37621 37151 MJECL31 37811 37599 MJECL32 40153 38828 MJECL33 41381 40125 MJECL34 43121 42231 MJECL35 45007 43115 MJECL36 45921 45394 MJECL37 46065 46865 MJECL38 47997 47197 MJECL39 49387 48329 MJECL41 53908 52613 MJECL43 57371 56187 MJECL44 58339 57341

TABLE 3 Genes of M. jannaschii that contain inteins. Gene No. of No. Putative identification inteins MJ0043 Hypothetical protein (Bacillus subtilis) 1 MJ0262 Putative translation initiation factor, FUN12/IF-2 1 family MJ0542 Phosphoenolpyruvate synthase 1 MJ0682 Hypothetical protein (Escherichia coli) 1 MJ0782 Tranascription initiation factor IIB 1 MJ0832 Anaerobic ribonucleoside-triphosphate reductase 2 MJ0885 DNA-dependent DNA polymerase, family B 2 MJ1042 DNA-dependent RNA polymerase, subunit A′ 1 MJ1043 DNA-dependent RNA polymerase, subunit A″ 1 MJ1054 UDP-glucose dehydrogenase 1 MJ1124 Hypothetical protein (Saccharomyces cerevisiae) 1 MJ1420 Glutamine-fructose-6-phosphate transaminase 1 MJ1422 Replication factor C, 37-kD subunit 3 MJ1512 Reverse gyrase 1

While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention.

All patents, patent applications and publications recited herein are hereby incorporated by reference.

SEQUENCE LISTING The patent contains a lengthy “Sequence Listing” section. A copy of the “Sequence Listing” is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/sequence.html?DocID=06797466B1). An electronic copy of the “Sequence Listing” will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3). 

What is claimed is:
 1. An isolated polynucleotide comprising the nucleic acid sequence of ORF MJ0428, represented by nucleotides 1087456-1088655 of SEQ ID NO:1.
 2. The isolated polynucleotide of claim 1, wherein said polynucleotide comprises a heterologous polynucleotide sequence.
 3. The isolated polynucleotide of claim 2, wherein said heterologous polynucleotide sequence encodes a heterologous polypeptide.
 4. A method for making a recombinant vector comprising inserting the isolated polynucleotide of claim 1 into a vector.
 5. A nucleic acid sequence complimentary to the polynucleotide of claim
 1. 6. A recombinant vector comprising the isolated polynucleotide of claim
 1. 7. The recombinant vector of claim 6, wherein said polynucleotide is operably associated with a heterologous regulatory sequence that controls gene expression.
 8. A recombinant host cell comprising the isolated polynucleotide of claim
 1. 9. The recombinant host cell of claim 8, wherein said polynucleotide is operably associated with a heterologous regulatory sequence that controls gene expression.
 10. An isolated polynucleotide fragment comprising a nucleic acid sequence which hybridizes under hybridization conditions, comprising hybridization in 5×SSC and 50% formamide at 50-65° C. and washing in a wash buffer consisting of 0.5×SSC at 50-65° C., to the complementary strand of ORF MJ0428, represented by nucleotides 1087456-1088655 of SEQ ID NO:1.
 11. The isolated polynucleotide of claim 10, wherein said polynucleotide comprises a heterologous polynucleotide sequence.
 12. The isolated polynucleotide of claim 11, wherein said heterologous polynucleotide sequence encodes a heterologous polypeptide.
 13. A method for making a recombinant vector comprising inserting the isolated polynucleotide of claim 10 into a vector.
 14. A nucleic acid sequence complimentary to the polynucleotide of claim
 10. 15. A recombinant vector comprising the isolated polynucleotide of claim
 10. 16. The recombinant vector of claim 15 wherein said polynucleotide is operably associated with a heterologous regulatory sequence that controls gene expression.
 17. A recombinant host cell comprising the isolated polynucleotide of claim
 10. 18. The recombinant host cell of claim 17, wherein said polynucleotide is operably associated with a heterologous regulatory sequence that controls gene expression.
 19. An isolated polynucleotide for the detection of Methanococcus jannaschii, wherein said isolated polynucleotide comprises at least 30 contiguous nucteotides of the nucleic acid sequence of ORF MJ0428, represented by nucleotides 1087456-1088655 of SEQ ID NO:1.
 20. The isolated polynucleotide of claim 19, wherein said polynucleotide comprises a heterologous polynucleotide sequence.
 21. The isolated polynucleotide of claim 20, wherein said heterologous polynucleotide sequence encodes a heterologous polypeptide.
 22. A method for making a recombinant vector comprising inserting the isolated polynucleotide of claim 19 into a vector.
 23. A nucleic acid sequence complimentary to the polynucleotide of claim
 19. 24. A recombinant vector comprising the isolated polynucleotide of claim
 19. 25. The recombinant vector of claim 24, wherein said polynucleotide is operably associated with a heterologous regulatory sequence that controls gene expression.
 26. A recombinant host cell comprising the isolated polynucleotide of claim
 19. 27. The recombinant host cell of claim 26, wherein said polynucleotide is operably associated with a heterologous regulatory sequence that controls gene expression.
 28. The isolated polynucleotide of claim 19, wherein said isolated polynucleotide comprises at least 40 contiguous nucleotides of the nucleic acid sequence of ORF MJ0428, represented by nucleotides 1087456-1088655 of SEQ ID NO:1.
 29. A method for detecting Methanococcus jannaschii comprising: (a) contacting a biological sample with the isolated polynucleotide of claim 10 under conditions suitable for the hybridization of said polynucleotide to a nucleic acid molecule complementary thereto; and (b) detecting the presence or absence of Methanococcus jannaschii in the biological sample.
 30. A method for detecting Methanococcus jannaschii comprising: (a) contacting said biological sample with the isolated polynucleotide of claim 19 under conditions suitable for the hybridization of said polynucleotide to a nucleic acid molecule complementary thereto; and (b) detecting the presence or absence of Methanococcus jannaschii in the biological sample.
 31. A method for detecting Methanococcus jannaschii comprising: (a) contacting said biological sample with the isolated polynucleotide of claim 28 under conditions suitable for the hybridization of said polynucleotide to a nucleic acid molecule complementary thereto; and (b) detecting the presence or absence of Methanococcus jannaschii in the biological sample. 